The Molecular Probes® Handbook A GUIDE TO FLUORESCENT … · 2 days ago · CHAPTER 16 Probes for Endocytosis, Receptors and Ion Channels Molecular Probes™ Handbook A Guide to

CHAPTER 16

Probes for Endocytosis, Receptors and Ion Channels

Molecular Probes™ HandbookA Guide to Fluorescent Probes and Labeling Technologies

11th Edition (2010)

CHAPTER 1

Fluorophores and Their Amine-Reactive Derivatives

The Molecular Probes® HandbookA GUIDE TO FLUORESCENT PROBES AND LABELING TECHNOLOGIES11th Edition (2010)

Molecular Probes® Resources

Molecular Probes® Handbook (online version)Comprehensive guide to �uorescent probes and labeling technologies

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Fluorescence SpectraViewerIdentify compatible sets of �uorescent dyes and cell structure probes

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BioProbes® Journal of Cell Biology ApplicationsAward-winning magazine highlighting cell biology products and applications

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Access all Molecular Probes® educational resources at lifetechnologies.com/mpeducate

Molecular Probes ResourcesMolecular Probes Handbook (online version)Comprehensive guide to fl uorescent probes and labeling technologiesthermofi sher.com/handbook

Molecular Probes Fluorescence SpectraViewerIdentify compatible sets of fl uorescent dyes and cell structure probesthermofi sher.com/spectraviewer

BioProbes Journal of Cell Biology ApplicationsAward-winning magazine highlighting cell biology products and applicationsthermofi sher.com/bioprobes

Access all Molecular Probes educational resources at thermofi sher.com/probes

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The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

SIX

TEEN

16.1 Probes for Following Receptor Binding and Phagocytosis. . . . . . . . . . . . . . . . . . . . . . . . . . . 741

Ligands for Studying Receptor-Mediated Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741

Fc OxyBURST® Green Assay Reagent: Fluorogenic Immune Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741

OxyBURST® Green H2HFF BSA Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742

Amine-Reactive OxyBURST® Green Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742

Fluorescent Low-Density Lipoprotein Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742

Fluorescent Acetylated LDL Complexes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 743

Fluorescent Lipopolysaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744

Epidermal Growth Factor Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744

Transferrin Conjugates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745

Fluorescent Fibrinogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746

DQ™ Ovalbumin: A Probe for Antigen Processing and Presentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747

Fluorescent Gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747

Fluorescent Casein. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747

Fluorescent Chemotactic Peptide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747

Fluorescent Insulin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748

Fluorescent Dexamethasone Probe for Glucocorticoid Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748

Fluorescent Histone H1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748

Fluorescent Probes for the Acrosome Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748

Methods for Detecting Internalized Fluorescent Ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 748

Membrane Markers of Endocytosis and Exocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 749

FM® 1-43. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 749

Other Analogs of FM® 1-43 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750

FM® 1-43FX and FM® 4-64FX: Fixable FM® Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750

4-Di-1-ASP and 4-Di-2-ASP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750

TMA-DPH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750

Fluorescent Cholera Toxin Subunit B: Markers of Lipid Rafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750

Fluorescent Protein–Based Lipid Raft Markers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751

Fluorescent Protein–Based Synaptic Vesicle Markers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751

Anti–Synapsin I Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752

High Molecular Weight Polar Markers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752

Fluorescent Protein–Based Endosomal Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752

BioParticles® Fluorescent Bacteria and Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753

Vybrant® Phagocytosis Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753

pHrodo™ BioParticles® Fluorescent Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 753

pHrodo™ Phagocytosis Particle Labeling Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754

Opsonizing Reagents and Non�uorescent BioParticles® Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754

Fluorescent Polystyrene Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754

Fluorescent Microspheres Coated with Collagen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754

Fluorescent Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755

pH Indicator Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755

CHAPTER 16

Probes for Endocytosis, Receptors and Ion Channels

The Molecular Probes™ Handbook: A Guide to Fluorescent Probes and Labeling Technologies

IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

thermofi sher.com/probes



Chapter 16 — Probes for Endocytosis, Receptors and Ion Channels

Low Molecular Weight Polar Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755

Data Table 16.1 Probes for Following Receptor Binding and Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 757

Product List 16.1 Probes for Following Receptor Binding and Phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 758

16.2 Probes for Neurotransmitter Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760

α-Bungarotoxin Probes for Nicotinic Acetylcholine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760

Fluorescent α-Bungarotoxins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760

Biotinylated α-Bungarotoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760

Unlabeled α-Bungarotoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761

Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761

BODIPY® FL Prazosin for α1-Adrenergic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 762

BODIPY® TMR-X Muscimol for GABAA Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 762

Fluorescent Angiotensin II for AT1 and AT2 Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 762

Naloxone Fluorescein for µ-Opioid Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763

Probes for Amino Acid Neurotransmitter Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763

Caged Amino Acid Neurotransmitters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763

Anti–NMDA Receptor Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763

Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763

Probes for Other Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 764

Data Table 16.2 Probes for Neurotransmitter Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765

Product List 16.2 Probes for Neurotransmitter Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765

16.3 Probes for Ion Channels and Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766

Probes for Ca2+ Channels and Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766

Fluorescent Dihydropyridine for L-Type Ca2+ Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766

BODIPY® FL Verapamil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766

Eosin Derivatives: Inhibitors of the Calcium Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767

Premo™ Cameleon Calcium Sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 767

Probes for Na+ Channels and Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768

Amiloride Analogs: Probes for the Na+ Channel and the Na+/H+ Antiporter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768

Ouabain Probes for Na+/K+-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768

Using BacMam Technology to Deliver and Express Sodium Channel cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768

Probes for K+ Channels and Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768

Glibenclamide Probes for the ATP-Dependent K+ Channel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768

FluxOR™ Potassium Ion Channel Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 769

Using BacMam Technology to Deliver and Express Potassium Channel cDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770

Probes for Anion Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770

Stilbene Disulfonates: Anion-Transport Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770

DiBAC4(5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771

Eosin Maleimide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771

Premo™ Halide Sensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 771

Data Table 16.3 Probes for Ion Channels and Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772

Product List 16.3 Probes for Ion Channels and Carriers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 772


IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes




Section 16.1 Probes for Following Receptor Binding and Phagocytosis

�e plasma membrane de�nes the inside and outside of the cell. It not only encloses the cytosol to maintain the intracellular environment but also serves as a formidable barrier to the extracellular environment. Because cells require input from their surroundings—in the form of hydrated ions, small polar molecules, large biomolecules and even other cells—they have developed strategies for overcoming this barrier. Some of these mechanisms involve initial formation of receptor–ligand complexes, followed by transport of the ligand across the cell membrane.1–5

�is section focuses on probes for following receptor binding, endocytosis and exocytosis. Section 16.2 describes tools for studying neurotransmitter receptors, which mediate external chemical messenger control over the electrical activity of neurons. Section 16.3 discusses strate-gies for monitoring ion channels and carriers, which are the molecular centerpiece of neural transmission and bioenergetics.

Ligands for Studying Receptor-Mediated EndocytosisWe o�er a variety of �uorescent and �uorogenic ligands that bind to membrane receptors

and are subsequently internalized. In some cases, the bound ligand is released intracellularly and the receptor is then recycled to the plasma membrane. Receptor binding may also result in signal transduction (Chapter 17), Ca2+ mobilization (Chapter 19), intracellular pH changes (Chapter 20) and formation of reactive oxygen species (ROS, Chapter 18).

Fc OxyBURST® Green Assay Reagent: Fluorogenic Immune ComplexWhen soluble or surface-bound IgG immune complexes interact with Fc receptors on phago-

cytic cells, a number of host defense mechanisms are activated, including phagocytosis and acti-vation of an NADPH oxidase–mediated oxidative burst.6 Dichlorodihydro�uorescein diacetate (H2DCFDA, D399; Section 18.2; Figure 16.1.1), a cell-permeant �uorogenic probe that localizes in the cytosol, has frequently been used to monitor this oxidative burst.7 Its �uorescence response, however, is limited by the di�usion rate of the reactive oxygen species into the cytosol from the phagovacuole where it is generated. In contrast, Fc OxyBURST® assay reagents permit direct measurement of the kinetics of Fc receptor–mediated internalization and the subsequent oxida-tive burst in the phagovacuole, yielding signals that are many times brighter than those generated by H2DCFDA (Figure 16.1.2, Figure 16.1.3).

Fc OxyBURST® Green assay reagent (F2902) was developed in collaboration with Elizabeth Simons of Boston University to monitor the oxidative burst in phagocytic cells using �uorescence instrumentation. Fc OxyBURST® Green assay reagent comprises bovine serum albumin (BSA) that has been covalently linked to dichlorodihydro�uorescein (H2DCF) and then complexed with a puri�ed rabbit polyclonal anti-BSA antibody (A11133). When these immune complexes bind to Fc receptors, the non�uorescent H2DCF molecules are internalized within the phago-vacuole and subsequently oxidized to green-�uorescent 2’,7’-dichloro�uorescein (DCF; Figure 16.1.2, Figure 16.1.3). Unlike H2DCFDA, Fc OxyBURST® Green assay reagent does not require intracellular esterases for activation, making this reagent particularly suitable for detecting the oxidative burst in cells with low esterase activity such as monocytes.8 Fc OxyBURST® Green assay reagent reportedly produces >8 times more �uorescence than does H2DCFDA at 60 seconds and >20 times more at 15 minutes following internalization of the immune complex.9

Published reports have described the use of Fc OxyBURST® Green assay reagent to study the oxidative burst in phagovacuoles.10–12 Neutrophils from patients with chronic granulomatous disease, a genetic de�ciency known to disable NADPH oxidase–mediated oxidative bursts, were

Figure 16.1.2 Fc OxyBURST® Green assay reagent (F2902) for �uorescent detection of the Fc receptor–mediated phagocytosis pathway. Dichlorodihydro�uorescein (H2DCF) is covalently attached to bovine serum albumin (BSA), then complexed with a rabbit polyclonal anti-BSA antibody (A11133). Upon binding to an Fc receptor, the non�uores-cent immune complex is internalized and subsequently oxi-dized to the �uorescent DCF.

Fc receptors

phagosome

H2DCF-BSAimmune complex

Figure 16.1.3 Fluorescence emission of human neutrophils challenged either with Fc OxyBURST® Green assay reagent (H2DCF-BSA immune complexes, F2902) or with unlabeled immune complexes in the presence of dichlorodihydro�uo-rescein diacetate (H2DCFDA; D399). Fc OxyBURST® Green as-say reagent generates signi�cantly more �uorescence than does the more commonly used H2DCFDA. Flow cytometry data provided by Elizabeth Simons, Boston University.

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H2DCF-BSAimmune complex

H2DCFDA

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Figure 16.1.1 2’,7’-dichlorodihydro�uorescein diacetate (2’,7’-dichloro�uorescin diacetate; H2DCFDA, D399).

16.1 Probes for Following Receptor Binding and Phagocytosis



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observed to bind but not oxidize Fc OxyBURST® Green assay reagent 9 (Figure 16.1.4). Using micro�uorometry to detect the Fc OxyBURST® Green signal, researchers were able to simulta-neously monitor oxidative activity and membrane currents in voltage-clamped human mono-nuclear cells.13

OxyBURST® Green H2HFF BSA ReagentOxyBURST® Green H2HFF BSA reagent 14–16 (O13291) is similar to Fc OxyBURST® Green

assay reagent, except that it is prepared by reacting the succinimidyl ester of a reduced form of our Oregon Green® 488 dye with BSA. �e absorption maximum of the oxidation product of this reagent (~492 nm) matches the 488 nm spectral line of the argon-ion laser better than does that of Fc OxyBURST® Green assay reagent (~495 nm). OxyBURST® Green H2HFF BSA reagent can also be complexed with anti-BSA antibody to form an immune complex that can be utilized like the Fc OxyBURST® Green assay reagent (F2902).

All of the OxyBURST® reagents are slowly oxidized by molecular oxygen and are also suscep-tible to oxidation catalyzed by illumination in a �uorescence microscope. �ese reagents are rea-sonably stable in solution for at least six months when stored under nitrogen or argon in the dark at 4°C. We also o�er a puri�ed rabbit polyclonal anti-BSA antibody (A11133), which can bind any of our BSA conjugates (Section 14.7) or �uorogenic DQ™ BSA conjugates (D12050, D12051; Section 10.4) to create immune complexes for analyzing the Fc receptor–mediated phagocyto-sis pathway. In the case of the anti-BSA antibody complex with DQ™ BSA, initial binding and internalization of the probe is followed by hydrolysis to �uorescent peptides by proteases in the phagovacuole 17 (Figure 16.1.5).

Amine-Reactive OxyBURST® Green ReagentAs an alternative to Fc OxyBURST® Green assay reagent and OxyBURST® Green H2HFF

BSA, we o�er amine-reactive OxyBURST® Green H2DCFDA succinimidyl ester (2’,7’-dichlo-rodihydro�uorescein diacetate, SE; D2935; Figure 16.1.6), which can be used to prepare oxi-dation-sensitive conjugates of a wide variety of biomolecules and particles, including antibod-ies, antigens, peptides, proteins, dextrans, bacteria, yeast and polystyrene microspheres.9,18,19 Following conjugation to amines, the two acetates of OxyBURST® Green H2DCFDA reagent can be removed by treatment with hydroxylamine at near-neutral pH to yield the oxidant-sensitive dichlorodihydro�uorescein conjugates. �us, like our Fc OxyBURST® Green assay reagent, they provide a means of detecting the oxidative burst in phagocytic cells.18

Several other reagents—dihydro�uoresceins, dihydrorhodamines, dihydroethidium and chemiluminescent probes—that have been used to detect the reactive oxygen species (ROS) pro-duced during phagocytosis are described in Section 18.2.

Fluorescent Low-Density Lipoprotein Complexes�e human LDL complex, which delivers cholesterol to cells by receptor-mediated endocy-

tosis, comprises a core of about 1500 molecules of cholesteryl ester and triglyceride, surrounded by a 20 Å–thick shell of phospholipids, unesteri�ed cholesterol and a single copy of apoprotein B 100 20 (MW ~500,000 daltons). Once internalized, LDL dissociates from its receptor and eventu-ally appears in lysosomes.21 In addition to unlabeled LDL (L3486), which has been reported to be an e�ective vehicle for selectively delivering antitumor drugs to cancer cells,22 we o�er two classes of labeled LDL probes—those containing an unmodi�ed apoprotein, used to study the mechanisms of normal cholesterol delivery and internalization, and those with an acetylated apoprotein, used to study endothelial, microglial and other cell types that express receptors that speci�cally bind this modi�ed LDL.

For the class of labeled LDL probes containing unmodi�ed apoprotein, we prepare LDL non-covalently labeled with either DiI (DiI LDL, L3482) or the BODIPY® FL �uorophore (BODIPY® FL LDL, L3483), highly �uorescent lipophilic dyes that di�use into the hydrophobic portion of the LDL complex without a�ecting the LDL-speci�c binding of the apoprotein. As compared with DiI LDL, BODIPY® FL LDL is more e�ciently excited by the 488 nm spectral line of the argon-ion laser, making it better suited for �ow cytometry and confocal laser-scanning micros-copy studies. Like our BODIPY® FL C5-ceramide (D3521, Section 12.4), BODIPY® FL LDL may exhibit concentration-dependent long-wavelength emission (>550 nm), precluding its use for

Figure 16.1.4 Oxidative bursts of human neutrophils from a healthy donor (control) compared with those from a patient with chronic granulomatous disease (CGD), as detected using the Fc OxyBURST® Green assay reagent (F2902). Flow cytom-etry data provided by Elizabeth Simons, Boston University.

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Figure 16.1.5 Immune complex of DQ™ BSA conjugate (D12050, D12051) with an anti–bovine serum albumin (BSA) antibody (A11133) for the �uorescent detection of the Fc re-ceptor–mediated phagocytosis pathway. The DQ™ BSA is a derivative of BSA that is labeled to such a high degree with either the green-�uorescent BODIPY® FL or red-�uorescent BODIPY® TR-X dye that the �uorescence is self-quenched. Upon binding to an Fc receptor, the non�uorescent immune complex is internalized and the protein is subsequently hy-drolyzed to �uorescent peptides within the phagovacuole.

Fc receptors

phagosome

DQ™ BSAimmune complex

Figure 16.1.6 2’,7’-dichlorodihydro�uorescein diacetate, suc-cinimidyl ester (OxyBURST® Green H2DCFDA, SE, D2935).







multicolor labeling with red �uorophores. Both DiI LDL and BODIPY® FL LDL have been used to investigate the binding speci�city and partitioning of LDL throughout the Schistosoma man-soni parasite 23 (Figure 16.1.7). Fluorescent LDL complexes have also proven useful in a variety of experimental systems to:

• Count the number of cell-surface LDL receptors, analyze their motion and clustering and follow their internalization 24–26

• Demonstrate that �broblasts grown continuously in the presence of DiI LDL (L3482) pro-liferate normally and exhibit normal morphology,27 making DiI LDL a valuable alternative to 125I-labeled LDL for quantitating LDL receptor activity 28

• Identify LDL receptor–de�cient Chinese hamster ovary (CHO) cell mutants 29

• Image LDL receptor endocytosis in COS7 cells expressing Green Fluorescent Protein (GFP)–tagged GTPase 30

• Investigate the modulation of LDL receptor expression by statin drugs 31,32

We prepare �uorescent LDL products from fresh human plasma, and they should be stored refrigerated and protected from light; LDL products must not be frozen. Because preparation of these complexes involves several variables, some batch-to-batch variability in degree of labeling and �uorescence yield is expected.

Fluorescent Acetylated LDL ComplexesIf the lysine residues of LDL’s apoprotein have been acetylated, the LDL complex no lon-

ger binds to the LDL receptor,33 but rather is taken up by macrophage and endothelial cells that possess “scavenger” receptors speci�c for the modi�ed LDL.34,35 Once the acetylated LDL (AcLDL) complexes accumulate within these cells, they assume an appearance similar to that of foam cells found in atherosclerotic plaques.36–38 We o�er unlabeled AcLDL (L35354), as well as AcLDL noncovalently labeled with DiI L(3484) and AcLDL covalently labeled with Alexa Fluor® 488 dye (L23380), Alexa Fluor® 594 dye (L35353) or BODIPY® FL dye (L3485). Fluorescent dye conjugates of high-density lipoproteins, including one prepared using Alexa Fluor® 488 suc-cinimidyl ester (A20000, A20100; Section 1.3), are taken up via the same receptor as acetylated LDL complexes.39

Using DiI AcLDL, researchers have discovered that the scavenger receptors on rabbit �-broblasts and smooth muscle cells appear to be up-regulated through activation of the protein kinase C pathway.40 DiI AcLDL has also been used to show that Chinese hamster ovary (CHO) cells express AcLDL receptors that are distinct from macrophage scavenger receptors.41,42 Ultrastructural localization of endocytic compartments that maintain a connection to the ex-tracellular space has been achieved by photoconversion of DiI AcLDL in the presence of diami-nobenzidine 43 (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2). A quantitative assay for LDL- and scavenger-receptor activity in adherent and nonad-herent cultured cells that avoids the use of both radioactivity and organic solvents has been described.44

It has now become routine to identify endothelial cells and microglial cells in primary cell culture by their ability to take up DiI AcLDL45,46 (Figure 16.1.8). DiI AcLDL was employed in order to con�rm endothelial cell identity in investigations of shear stress 47 and P-glycoprotein expression,48 as well as to identify blood vessels in a growing murine melanoma.49 In addition, patch-clamp techniques have been used to investigate membrane currents in mouse microglia, which were identi�ed both in culture and in brain slices by their staining with DiI AcLDL.50,51 For some applications, Alexa Fluor® 488, Alexa Fluor® 594 and BODIPY® FL AcLDL may be the preferred probes because the dyes are covalently bound to the modi�ed apoprotein portion of the LDL complex and are therefore not extracted during subsequent manipulations of the cells. Furthermore, the green-�uorescent Alexa Fluor® 488 AcLDL has spectral characteristics similar to �uorescein and is useful for analyses with instruments equipped with the 488 nm argon-ion laser excitation sources, including �ow cytometers and confocal laser-scanning microscopes. �e bright and photostable red-�uorescent Alexa Fluor® 594 AcLDL conjugate is useful for multila-beling experiments with green-�uorescent probes and can be e�ciently excited by the 594 nm spectral line of the orange He-Ne laser.52

Figure 16.1.7 DiI LDL (L3482) bound to the surface and internalized in the gut of the parasite Schistosoma mansoni. The distribution of LDL in the parasite is used to study a pu-tative mechanism by which the parasite may avoid host im-mune recognition. Image contributed by John P. Caul�eld, Harvard School of Public Health.

Figure 16.1.8 Microglial cells in a rat hippocampus cryo-section labeled with red-orange–�uorescent DiI acetylated low-density lipoprotein (L3484) and stained using blue-�u-orescent DAPI (D1306, D3571, D21490).








Fluorescent LipopolysaccharidesWe o�er �uorescent conjugates of lipopolysaccharides (LPS) from Escherichia coli and

Salmonella minnesota (Table 16.1), including:

• Alexa Fluor® 488 LPS from E. coli serotype 055:B5 (A23351)• Alexa Fluor® 488 LPS from S. minnesota (A23356)• Alexa Fluor® 568 LPS from E. coli serotype 055:B5 (A23352)• Alexa Fluor® 594 LPS from E. coli serotype 055:B5 (A23353)• BODIPY® FL LPS from E. coli serotype 055:B5 (A23350)

LPS, also known as endotoxins, are a family of complex glycolipid molecules located on the surface of gram-negative bacteria. LPS play a large role in protecting the bacterium from host defense mechanisms and antibiotics. Binding of LPS to the CD14 cell-surface receptor of phago-cytes is the key initiation step in the mammalian immune response to infection by gram-negative bacteria.53 �e structural core of LPS, and the primary determinant of its biological activity, is an N-acetylglucosamine derivative, lipid A (Figure 16.1.9). In many gram-negative bacterial infec-tions, LPS are responsible for clinically signi�cant symptoms like fever, low blood pressure and tissue edema, which can lead to disseminated intravascular coagulation, organ failure and death.

�e �uorescent BODIPY® FL and Alexa Fluor® LPS conjugates, which are labeled with suc-cinimidyl esters of these dyes, allow researchers to follow LPS-elicited in�ammatory respons-es.53,54 Lipopolysaccharide internalization activates endotoxin-dependent signal transduction in cardiomyocytes.55 Alexa Fluor® 488 LPS conjugates (L23351, L23356) have been shown to selectively label microglia in a mixed culture containing oligodendrocyte precursors, astrocytes and microglia.56

�e BODIPY® FL derivative of LPS from E. coli strain LCD25 (L23350) was used to measure the transfer rate of LPS from monocytes to high-density lipoprotein 57 (HDL). Another study utilized a BODIPY® FL derivative of LPS from S. minnesota to demonstrate transport to the Golgi apparatus in neutrophils,58,59 although this could have been due to probe metabolism. It has been reported that organelles other than the Golgi are labeled by some �uorescent or non�uorescent LPS.60,61 Cationic lipids are reported to assist the translocation of �uorescent lipopolysaccharides into live cells; 62 cell surface–bound LPS can be quenched by trypan blue.57

Other probes useful for analyzing lipopolysaccharides include �uorescent analogs of the LPS-binding antibiotic polymyxin B (Section 17.3) and BODIPY® TR cadaverine (D6251, Section 3.4). BODIPY® TR cadaverine binds with high selectivity to lipid A, forming the basis for high-throughput ligand displacement assays for identifying endotoxin antagonists.63,64

Epidermal Growth Factor DerivativesEpidermal growth factor (EGF) is a 53–amino acid polypeptide hormone (MW 6045 daltons)

that stimulates division of epidermal and other cells. �e EGF receptors include the HER-2/neu receptor (where “HER-2” is an acronym for human epidermal growth factor receptor-2 and “neu” refers to an original mouse origin); HER-2/neu overexpression has evolved as a prognostic/predictive factor in some solid tumors.65–67 Binding of EGF to its 170,000-dalton receptor pro-tein results in the activation of kinases, phospholipases and Ca2+ mobilization and precipitates a wide variety of cellular responses related to di�erentiation, mitogenesis, organ development and cell motility.

We o�er unlabeled mouse submaxillary gland EGF (E3476), as well as the following EGF conjugates, each containing a single �uorophore or biotin on the N-terminal amino acid:

• Fluorescein EGF (E3478)• Oregon Green® 514 EGF (E7498)• Tetramethylrhodamine EGF (E3481)• Biotin-XX EGF (E3477)

�e dissociation constant of the EGF conjugates in DMEM-HEPES medium is in the low nanomolar range for human epidermoid carcinoma (A431) cells,68 a value that approximates that of the unlabeled EGF. Fluorescently labeled EGF has enabled scientists to use �uorescence resonance energy transfer techniques to assess EGF receptor–receptor and receptor–membrane interactions 69–71 (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). Using �uorescein

Figure 16.1.9 Structure of the lipid A component of Salmonella minnesota lipopolysaccharide.

O

OH

HO P O

OH

HO

O O

O

O

O

O

NH

O

HO

O

O

O

O

O

NH O P

O

OH

OH

O

O

O

Figure 16.1.10 Detection of epidermal growth factor (EGF) receptors directly or with signal ampli�cation. Cells express-ing high (A431 cells, panel A) and low (NIH 3T3 cells, panel B) levels of EGF receptors were either directly labeled with the preformed Alexa Fluor® 488 complex of biotinylated epidermal growth factor (E13345, blue) or indirectly labeled with biotinylated EGF (E3477) followed by either Alexa Fluor® 488 streptavidin (S11223, green) or HRP-conjugated streptavidin and Alexa Fluor® 488 tyramide (purple), compo-nents of our TSA™ Kit #22 (T20932).

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Table 16.1 Fluorescent lipopolysaccharide conjugates.

Fluorophore Abs * Em * Escherichia coli Salmonella minnesota

Alexa Fluor® 488 495 519 L23351 L23356

BODIPY® FL 503 513 † L23350

Alexa Fluor® 568 578 603 L23352

Alexa Fluor® 594 590 617 L23353

* Approximate absorption (Abs) and �uorescence emission (Em) maxima for conjugates, in nm. † At high concentrations, the emission maximum for the BODIPY® FL dye may shift from ~513 nm to ~620 nm.1,2

1. J Immunol (1997) 158:3925; 2. J Biol Chem (1996) 271:4100.

EGF as the donor and tetramethylrhodamine EGF as the acceptor, researchers examined tem-perature-dependent lateral and transverse distribution of EGF receptors in A431 cell plasma membranes.71 When �uorescein EGF binds to A431 cells, it apparently undergoes a biphasic quenching, which can be attributed �rst to changes in rotational mobility upon binding and then to receptor–ligand internalization. By monitoring this quenching in real time, the rate constants for the interaction of �uorescein EGF with its receptor were determined.72 Although �uorescently labeled EGF can be used to follow lateral mobility and endocytosis of the EGF receptor,73,74 the visualization of �uorescent EGF may require low-light imaging technology or Qdot® nanocrystals, especially in cells that express low levels of the EGF receptor.75 In cells with few EGF receptors, it can be di�cult to detect signal over background �uorescence unless signal ampli�cation methods are employed (Figure 16.1.10).

Biotin-XX EGF contains a long spacer arm that enhances the probe’s a�nity for the EGF receptor and facilitates binding of dye-, Qdot® nanocrystal– or enzyme-conjugated streptavi-dins 75–79 (Section 7.6). Using biotinylated EGF and phycoerythrin-labeled secondary reagents (Section 6.4), researchers were able to detect as few as 10,000 EGF cell-surface receptors by confo-cal laser-scanning microscopy.80 Tyramide signal ampli�cation (TSA) technology (Section 6.2) is particularly valuable for detection and localization of low-abundance EGF receptors by both imaging and �ow cytometry (Figure 16.1.10). For additional sensitivity, we prepare biotinylated EGF precomplexed to �uorescent streptavidin:

• Biotinylated EGF complexed to Alexa Fluor® 488 streptavidin (E13345, Figure 16.1.11)• Biotinylated EGF complexed to Alexa Fluor® 555 streptavidin (E35350)• Biotinylated EGF complexed to Alexa Fluor® 647 streptavidin (E35351)• Biotinylated EGF complexed to Texas Red® streptavidin 81,82 (E3480)

�ese products yield several-fold brighter signals per EGF receptor when compared with the direct conjugates. We have found that EGF receptors can easily be detected with these complexes without resorting to low-light imaging technology (Figure 16.1.12). A quantitative high-content screening (HCS) assay for EGF receptor modulators based on imaging the internalization of the Alexa Fluor® 555 EGF complex internalization has been reported.83

Transferrin ConjugatesTransferrin is a monomeric serum glycoprotein (MW ~80,000 daltons) that binds up to two

Fe3+ atoms for delivery to vertebrate cells through receptor-mediated endocytosis. Once iron-carrying transferrin proteins are inside endosomes, the acidic environment favors dissociation of the sequestered iron from the transferrin–receptor complex. Following the release of iron, the apotransferrin is recycled to the plasma membrane, where it is released from its receptor to scavenge more iron. Transferrin uptake is a prototypical and ubiquitous example of clathrin-mediated endocytosis. Although transferrin uptake is widely regarded as a surrogate measure of total clathrin-mediated endocytosis, perturbations that are speci�c to transferrin endocytosis impel caution in making such extrapolations.2

Our �uorescent and biotinylated di-ferric (Fe3+) human transferrin conjugates (Table 16.2) include:

• Fluorescein transferrin (T2871)• Alexa Fluor® 488 transferrin 84–87 (T13342)• Alexa Fluor® 546 transferrin 88,89 (T23364)• Alexa Fluor® 555 transferrin 90 (T35352)

Figure 16.1.12 Lightly �xed human epidermoid carcino-ma cells (A431) stained with biotinylated epidermal growth factor (EGF) complexed to Texas Red® streptavidin (E3480). An identical cell preparation stained in the presence of a 100-fold excess of unlabeled EGF (E3476) showed no �uo-rescent signal.

Figure 16.1.11 Early endosomes in live HeLa cells identi-�ed after a 10-minute incubation with green-�uorescent Alexa Fluor® 488 epidermal growth factor (E13345). The cells were subsequently �xed with formaldehyde and la-beled with an antibody to the late endosomal protein, RhoB. That antibody was visualized with a red-orange–�uorescent secondary antibody. Nuclei were stained with TO-PRO®-3 iodide (T3605, pseudocolored blue). The image was contrib-uted by Harry Mellor, University of Bristol.

Table 16.2 Transferrin conjugates.

Cat. No. Label Abs * Em *

T2871 Fluorescein 494 518

T13342 Alexa Fluor® 488 495 518

T2872 Tetramethylrhodamine 555 580

T23364 Alexa Fluor® 546 556 575

T35352 Alexa Fluor® 555 555 565

T23365 Alexa Fluor® 568 578 603

T13343 Alexa Fluor® 594 589 616

T2875 Texas Red® 595 615

T23362 Alexa Fluor® 633 632 647

T23366 Alexa Fluor® 647 650 665

T35357 Alexa Fluor® 680 679 702

T23363 Biotin-XX NA NA

* Approximate absorption (Abs) and �uorescence emission (Em) maxima for conjugates, in nm. NA = Not applicable.








• Alexa Fluor® 568 transferrin 91 (T23365)• Alexa Fluor® 594 transferrin 92–94 (T13343, Figure 16.1.13)• Alexa Fluor® 633 transferrin (T23362)• Alexa Fluor® 647 transferrin 73,95,96 (T23366)• Alexa Fluor® 680 transferrin (T35357)• Tetramethylrhodamine transferrin (T2872)• Texas Red® transferrin (T2875)• Biotin-XX transferrin (T23363)

Alexa Fluor® transferrin conjugates are highly recommended because of their brightness, enhanced photostability and lack of sensitivity to pH (Section 1.3). �e pH sensitivity of �uo-rescein-labeled transferrin has been exploited to investigate events occurring during endosomal acidi�cation.97–100 Fluorescent transferrins have also been used to:

• Analyze the role of the γ-chain of type III IgG receptors in antigen–antibody complex internalization 101

• Characterize endocytic apparatus phenotypes in drug-resistant cancer cells 85

• Demonstrate that the fungal metabolite brefeldin A (B7450, Section 12.4) induces an increase in tubulation of transferrin receptors in BHK-21 cells 102 and in the perikaryal–dendritic region of cultured hippocampal neurons 103

• Image transferrin receptor dynamics using FRET 104

• Observe receptor tra�cking in live cells by confocal laser-scanning microscopy 74

Uptake of a horseradish peroxidase (HRP) conjugate of transferrin by endosomes has been detected using tyramide signal ampli�cation (TSA, Section 6.2) by catalytic deposition of biotin tyramide and use of �uorescent streptavidin conjugates 105 (Section 7.6).

In addition to �uorescent and biotinylated transferrin conjugates, we o�er a mouse monoclo-nal IgG1 anti–human transferrin receptor antibody (A11130). �is antibody can be used with any of our Zenon® Mouse IgG1 Labeling Kits (Section 7.3, Table 7.7) for rapid preparation of labeling complexes. Antibodies against transferrin receptors have been used for indirect immuno�uo-rescent staining of the transferrin receptor,106–108 transport of molecules across the blood–brain barrier,109 characterization of transferrin in recycling compartments,106 enzyme-linked immu-nosorbent assays (ELISAs) 108 and antibody competition with transferrin uptake.110

Fluorescent FibrinogenFibrinogen is a key component in the blood clotting process and can support both platelet–

platelet and platelet–surface interactions by binding to the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor (also called integrin αIIbβ3) of activated platelets. Activation of GPIIb-IIIa is required for �brinogen binding, which leads to platelet activation, adhesion, spreading and micro�lament reorganization of human endothelial cells in vitro. Bone marrow transplant patients have signi�-cantly higher levels of �brinogen binding, as compared with controls Soluble �brinogen binds to its receptor with a Ca2+-dependent apparent Kd of 0.18 µM.111 �is binding is mediated by the tripeptide sequence Arg-Gly-Asp (RGD), found in both �brinogen and �bronectin.

Fluorescently labeled �brinogen has proven to be a valuable tool for investigating platelet activa-tion and subsequent �brinogen binding.112–114 Alexa Fluor® 647 �brinogen has been used to identify activated platelets by �ow cytometry.115 �e binding of �uorescein �brinogen to activated platelets has been shown to be saturable and can be inhibited completely by underivatized �brinogen.116,117

We o�er four conjugates of human �brinogen in three di�erent �uorescent colors:

• Alexa Fluor® 488 human �brinogen conjugate (F13191)• Oregon Green® 488 human �brinogen conjugate (F7496)• Alexa Fluor® 546 human �brinogen conjugate (F13192)• Alexa Fluor® 647 human �brinogen conjugate (F35200)

�ese highly �uorescent �brinogen conjugates are useful for investigating platelet activation and subsequent �brinogen binding using �uorescence microscopy or �ow cytometry 112,115,118 (Figure 16.1.14).

Figure 16.1.14 Interaction of �uorescently labeled �brino-gen with activated platelets. Whole blood was �rst incubat-ed with an R-phycoerythrin (R-PE)–labeled anti-CD41 anti-body to label the platelets. 20 µM adenosine 5’-diphosphate (ADP) was added in order to activate the platelets, then 2 µg/mL Alexa Fluor® 488 �brinogen (F13191) was added and incubated with the sample for 5 minutes. Cells were an-alyzed by �ow cytometry using excitation at 488 nm. Both activated and unactivated platelets show binding of the anti-CD41 antibody; however, only the activated platelets show strong binding by �brinogen. A total of 5000 platelets are shown in each experiment.

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Unactivated

Activated

Figure 16.1.13 Live HeLa cells incubated with Alexa Fluor® 594 transferrin (T13343) for 10 minutes to label early endo-somes. The cells were subsequently �xed with formaldehyde and labeled with an antibody to the endosomal protein RhoD. That antibody was visualized with a green-�uo rescent second-ary antibody. Yellow �uorescence indicates regions of co-local-ization. To illustrate the staining pattern, the cells were imaged by both �uorescence (top panel) and di�erential interference contrast (DIC) microscopy (bottom panel). The image was con-tributed by Harry Mellor, University of Bristol.







DQ™ Ovalbumin: A Probe for Antigen Processing and Presentation

Although antigen processing and presentation have been exten-sively studied, the exact sequence and detailed pathways for generating antigenic peptides have yet to be elucidated. In general, the immuno-genic protein is internalized by a macrophage, denatured, reduced and proteolyzed, and then the resulting peptides associate with MHC class II molecules that are expressed at the cell surface.119 Ovalbumin is ef-�ciently processed through mannose receptor–mediated endocytosis by antigen-presenting cells and is widely used for studying antigen processing.120–122 DQ™ ovalbumin 123 (D12053), a self-quenched oval-bumin conjugate, is designed speci�cally for the study of macrophage-mediated antigen processing in �ow cytometry and microscopy assays.

Traditionally, �uorescein-labeled bovine serum albumin (FITC-BSA) has been used as a �uorogenic protein antigen for studying the real-time kinetics of antigen processing in live macrophages by �ow cytometry,124 two-photon �uorescence lifetime imaging microscopy (FLIM) 125 and �uorescence polarization.124,126,127 FITC-ovalbumin has been employed to study antigen uptake in HIV-1–infected monocytic cells.128 �e FITC-ovalbumin and FITC-BSA used in these experiments were heavily labeled with �uorescein such that the intact conjugates were relatively non�uorescent due to self-quenching. Upon denatur-ation and proteolysis, however, these FITC conjugates became highly �uorescent, allowing researchers to monitor intracellular tra�cking and the processing of ovalbumin and BSA in macrophages.

For studies of antigen processing and presentation, DQ™ ovalbumin o�ers several advantages when compared with FITC-ovalbumin and FITC-BSA. Like the FITC conjugates, DQ™ ovalbumin is labeled with our pH-insensitive, green-�uorescent BODIPY® FL dye such that the �uorescence is almost completely quenched until the probe is digested by proteases (Figure 16.1.15). Unlike �uorescein, which has greatly re-duced �uorescence intensity at acidic pH and is not very photostable, our BODIPY® FL dye exhibits bright, relatively photostable and pH-in-sensitive �uorescence from pH 3 to 9. Furthermore, the intact DQ™ ov-albumin is more highly quenched than unprocessed FITC-ovalbumin or FITC-BSA at a lower degree of substitution, thereby providing a lower background signal while preserving the protein’s antigenic epi-topes. Although we o�er the green-�uorescent DQ™ Green BSA and red-�uorescent DQ™ Red BSA (D12050, D12051; Section 10.4), which are also self-quenched BODIPY® FL and BODIPY® TR conjugates, we highly recommend DQ™ ovalbumin (D12053) for studying antigen processing and presentation 129,130 because ovalbumin is internalized via the mannose receptor–mediated endocytosis pathway and is thus processed more e�ciently by antigen-presenting cells than is BSA.131

Fluorescent GelatinCollagen is a major component of the extracellular matrix and, in

vertebrates, constitutes approximately 25% of total protein. �is im-portant protein not only serves a structural role, but also is important in cell adhesion and migration. Speci�c collagen receptors, �bronectin and a number of other proteins involved in cell–cell and cell–surface adhesion have been demonstrated to bind collagen and gelatin 132,133 (denatured collagen).

We o�er highly �uorescent gelatin conjugates for researchers study-ing collagen-binding proteins and collagen metabolism, as well as gelati-nases and collagenases, which are metalloproteins that digest gelatin and collagen. We o�er two green-�uorescent gelatin conjugates—�uorescein

Figure 16.1.15 Principle of enzyme detection via the disruption of intramolecular self-quenching. Enzyme-catalyzed hydrolysis of the heavily labeled and almost totally quenched substrates provided in our EnzChek® Protease Assay Kits (E6638, E6639), EnzChek® Ultra Amylase Assay Kit (E33651), EnzChek® Gelatinase/Collagenase Assay Kit (E12055), EnzChek® Elastase Kit (E12056), EnzChek® Lysozyme Assay Kit (E22013)—as well as the stand-alone quenched substrates DQ™ BSA (D12050, D12051), DQ™ collagen (D12052, D12060), DQ™ ov-albumin (D12053) and DQ™ gelatin (D12054)—relieves the intramolecular self-quenching, yielding brightly �uorescent reaction products.

Intramolecularlyquenched substrate

Enzyme

Fluorescent cleavage products

gelatin and Oregon Green® 488 gelatin (G13187, G13186). Fluorescent gelatin conjugates have been shown to be useful for:

• Assessing gelatinase activity in podosomes of mouse dendritic cells 134

• Localizing surface �bronectin on cultured cells 135

• Performing in situ gelatinase zymography on canary brain sections 136

• Studying �bronectin–gelatin interactions in solution using �uores-cence polarization 133 (Fluorescence Polarization (FP)—Note 1.4)

We have also developed �uorogenic gelatinase and collagenase substrates—DQ™ gelatin and DQ™ collagen (Figure 16.1.15) (D12054, D12060)—that are described in Section 10.4. In addition, we o�er �uo-rescent microspheres coated with collagen, which are described below.

Fluorescent CaseinReal-time imaging of �uorescein-labeled casein (C2990) and

FluoSpheres® �uorescent microspheres has been used to character-ize the endocytic apparatus of the protozoan Giardia lamblia.137 �e EnzChek® Protease Assay Kits (E6638, E6639; Section 10.4) provide convenient �uorescence-based assays for protease activity and con-tain either green-�uorescent BODIPY® FL casein or red-�uorescent BODIPY® TR-X casein 138 (Figure 16.1.15). BODIPY® FL casein and BODIPY® TR-X casein have signi�cant utility as nontoxic and pH-insensitive general markers for phagocytic cells in culture.139,140 Our RediPlate™ 96 (R22132) version of the BODIPY® TR-X casein substrate (Section 10.4) is ideal for high-throughput screening of potential pro-tease inhibitors.

Fluorescent Chemotactic PeptideA variety of white blood cells containing the formyl-Met-Leu-Phe

(fMLF) receptor respond to bacterial N-formyl peptides by migrating to the site of bacterial invasion and then initiating an activation pathway to control the spread of infection. Activation involves Ca2+ mobiliza-tion,141 transient acidi�cation,142,143 actin polymerization,144 phagocyto-sis 145 and production of oxidative species.146 We o�er the �uorescein conjugate of the hexapeptide formyl-Nle-Leu-Phe-Nle-Tyr-Lys (F1314),








which has been extensively employed to investigate the fMLF receptor.147–151 �e �uorescein-la-beled chemotactic peptide has been used to study G-protein coupling and receptor structure,152–154 expression,155,156 distribution 157–159 and internalization.160

Fluorescent InsulinWe prepare a high-purity, zinc-free �uorescein isothiocyanate conjugate of human insulin

(FITC insulin, I13269). Unlike most commercially available preparations, our FITC insulin is puri-�ed by HPLC and consists of a singly labeled species of insulin that has been speci�cally modi�ed at the N-terminus of the B-chain. Because the degree and position of labeling can alter the biologi-cal activity of insulin, we have isolated the singly labeled species that has been shown to retain its biological activity in an autophosphorylation assay.161 Our FITC insulin preparation is useful for imaging insulin and insulin receptor distribution,162 as well as for conducting insulin-binding as-says using micro�uidic devices.163,164

Fluorescent Dexamethasone Probe for Glucocorticoid Receptors�e synthetic steroid hormone dexamethasone binds to the glucocorticoid receptor, produc-

ing a steroid–receptor complex that then localizes in the nucleus and regulates gene transcription. In hepatoma tissue culture (HTC) cells, tetramethylrhodamine-labeled dexamethasone has been shown to have high a�nity for the glucocorticoid receptor in a cell-free system and to induce tyro-sine aminotransferase (TAT) expression in whole cells, albeit at a much lower rate than unmodi�ed dexamethasone.165 �is labeled dexamethasone also allowed the �rst observations of the �uorescent steroid–receptor complex in the HTC cell cytosol.165 Fluorescein dexamethasone (D1383, Figure 16.1.16) should be similarly useful for studying the mechanism of glucocorticoid receptor activation.

Fluorescent Histone H1�e Alexa Fluor® 488 conjugate of the lysine-rich calf thymus histone H1 (H13188) is a useful

probe for nuclear protein transport assays.166 Nuclear-to-mitochondrial translocation of histone H1 is indicative of dsDNA strand breaks. Fluorescent histone H1 conjugates can also be used to detect membrane-surface exposure of acidic phospholipids such as phosphatidylserine.167

Fluorescent Probes for the Acrosome ReactionSoybean trypsin inhibitor (SBTI) inhibits the catalytic activity of serine proteases and binds

to acrosin, an acrosomal serine protease associated with binding of spermatozoa to the zona pellucida.168 Alexa Fluor® 488 dye–labeled trypsin inhibitor from soybean (T23011) is useful for real-time imaging of the acrosome reaction in live spermatozoa.169 A �uorescent peanut lectin has been combined with ethidium homodimer-1 (EthD-1, E1169; Section 15.2) for a combined acrosome reaction assay and vital staining.170 Alexa Fluor® 488, Alexa Fluor® 568, Alexa Fluor® 594 and Alexa Fluor® 647 conjugates of Arachis hypogaea lectin (PNA) (L21409, L32458, L32459, L32460) have similar utility as acrosomal stains.171

Methods for Detecting Internalized Fluorescent LigandsMany of the �uorescent ligands described in this section �rst bind to cell-surface receptors,

then are internalized and, in some cases, recycled to the cell surface. In most applications, the cell-surface and internalized ligand populations are spatially resolved by imaging. It is o�en desirable to include noninternalized plasma membrane reference markers in these labeling pro-tocols. CellMask™ Orange and CellMask™ Deep Red plasma membrane stains (C10045, C10046; Section 14.4) are particularly suitable for this purpose.172,173 Other useful membrane markers in-clude posttranslationally lipidated �uorescent proteins 174 (O36214, O10139; Section 14.4). When spatial resolution is not possible, there are other means by which these signals can be separated and, in some cases, quantitated. �ese include:

• Use of antibodies to the Alexa Fluor® 488, BODIPY® FL, �uorescein/Oregon Green®, tetramethylrhodamine, Texas Red® and Alexa Fluor® 405/Cascade Blue® dyes (Section 7.4, Table 7.8) to quench most of the �uorescence of surface-bound or exocytosed probes

Figure 16.1.16 Dexamethasone �uorescein (D1383).

Figure 16.1.17 Principle of the Vybrant® Phagocytosis Assay Kit (V6694) for the simple quantitation of phagocyto-sis. A) Brie�y, phagocytic cells are incubated with the green-�uorescent �uorescein-labeled Escherichia coli BioParticles® conjugates (E2861). B) The �uorescence from any noninter-nalized BioParticles® product is then quenched by the ad-dition of trypan blue, and the samples are subsequently as-sayed with a �uorescence microplate reader equipped with �lters for the detection of �uorescein (FITC).

Trypan blue

Figure 16.1.18 Tracking endocytosis inhibition with pHrodo™ dextran conjugates. HeLa cells were plated in 96-well format and treated with dynasore for 3 hours at 37°C prior to the pHrodo™ endocytosis assay. Next, 40 µg/mL of pHrodo™ 10,000 MW dextran (P10361) was incubated for 30 minutes at 37°C, and cells were then stained with HCS NuclearMask™ Blue Stain (H10325) for 10 minutes to reveal total cell number and demarcation for image analy-sis. Images were acquired on the BD Pathway™ 855 High-Content Bioimager (BD Biosciences).

[Dynasore] (M)10–9 10–510–610–710–8 10–4

0.5

Nor

mal

ized

�uo

resc

ence

1.0

1.5

2.0

2.5

3.0IC50 = 4.7 µM

A

B







• Use of a dye such as trypan blue to quench external �uorescent signals but not internalized signals 175,176 (Figure 16.1.17)—a method employed in our Vybrant® Phagocytosis Assay Kit (V6694) described below

• Rapid acidi�cation of the medium to quench the �uorescence of pH-sensitive �uorophores such as �uorescein on the cell surface, thus enabling selective detection of endocytosed probe

• Tagging of proteins, polysaccharides, cells, bacteria, yeast, fungi 177 and other materials to be endocytosed with a pH-sensitive dye—such as our pHrodo™,178–180 SNARF® or Oregon Green® dyes (Chapter 20)—that undergoes a spectral shi� or intensity change in the acidic pH range found in phagovacuoles and late endosomes

• Use of heavily labeled, highly quenched proteins such as our DQ™ BSA and DQ™ gelatin probes, which yield highly �uorescent peptides upon intracellular proteolysis 181 (Section 10.4)

Pathway-speci�c inhibitors—such as chloropromazine, dynasore (Figure 16.1.18), dansyl cada verine (D113, Section 3.4), brefeldin A (B7450, Section 12.4), genistein and �lipin—are widely used in combination with �uorescently labeled ligands for characterizing endocytic pathways.182 A critical evaluation 183 highlights some necessary cautions in the application and interpretation of this approach, relating to decreased cell viability caused by some inhibitors as well as cell type–dependent di�erences in their e�cacy.

Membrane Markers of Endocytosis and ExocytosisFM® 1-43

FM® dyes—FM® 1-43, FM® 2-10, FM® 4-64, FM® 5-95 and the aldehyde-�xable FM® 1-43FX and FM® 4-64FX—are excellent membrane probes both for identifying actively �ring neurons 184 and for investigating the mechanisms of activity-dependent vesicle cycling in widely di�erent species.185–188 FM® dyes may also be useful as general-purpose probes for investigating endocy-tosis and for simply identifying cell membrane boundaries.

FM® 1-43 and its analogs, which are nontoxic to cells and virtually non�uorescent in aque-ous medium, are believed to insert into the outer lea�et of the surface membrane, where they become intensely �uorescent. In a neuron that is actively releasing neurotransmitters, these dyes become internalized within the recycled synaptic vesicles and the nerve terminals become brightly stained (Figure 16.1.19, Figure 16.1.20). �e nonspeci�c staining of cell-surface mem-branes can simply be washed o� prior to viewing. Wash removal of noninternalized dye back-ground is more di�cult in tissue preparations than in disseminated cell cultures. Extracellular �uorescence quenching 189 and dye adsorption 190 strategies have been developed to address this problem. Alternatively, the optical sectioning capabilities of confocal microscopy, two-photon excitation microscopy (Fluorescent Probes for Two-Photon Microscopy—Note 1.5) and total internal re�ection (TIRF) microscopy provide instrument-based solutions for improving the signal-to-background contrast.191 �e amount of FM® 1-43 taken up per vesicle by endocytosis equals the amount of dye released upon exocytosis, indicating that the dye does not transfer from internalized vesicles to an endosome-like compartment during the recycling process.192 In astrocytes, internalization of FM® 1-43 (and FM® 4-64) is mediated by store-operated calcium channels and not by endocytosis.193 Like most styryl dyes, the absorption and �uorescence emission spectra of FM® 1-43 are signi�cantly shi�ed in the membrane environment and are relatively broad (Figure 16.1.21), requiring careful matching with other �uorophores to avoid channel crosstalk in multiplex detection applications (Using the Fluorescence SpectraViewer—Note 23.1). We o�er FM® 1-43 in a 1 mg vial (T3163) or specially packaged in 10 vials of 100 µg each (T35356).

FM® 1-43 was employed in a study showing that synaptosomal endocytosis is independent of both extracellular Ca2+ and membrane potential in dissociated hippocampal neurons,194 as well as in a spectro�uorometric assay demonstrating that nitric oxide–stimulated vesicle re-lease is independent of Ca2+ in isolated rat hippocampal nerve terminals.195 FM® 1-43 has been used in combination with fura-2 (Section 19.2) to simultaneously measure intracellular Ca2+ and membrane turnover.196,197 FM® 1-43 dye–mediated photoconversion has been used to visualize recycling vesicles in hippocampal neurons.198

Figure 16.1.19 Live nerve terminals of motor neurons that innervate a rat lumbrical muscle stained with the activity-dependent dye FM® 1-43 (T3163, T35356) and observed under low magni�cation. The dye molecules, which insert into the outer lea�et of the surface membrane, are cap-tured in recycled synaptic vesicles of actively �ring neurons. The image was contributed by William J. Betz, University of Colorado School of Medicine.

Figure 16.1.21 Absorption and �uorescence emission spec-tra of FM® 1-43 bound to phospholipid bilayer membranes.

Figure 16.1.20 A feline mesenteric Pacinian corpuscle la-beled with FM® 1-43 (T3163, T35356). The image was con-tributed by Michael Chua, University of North Carolina at Chapel Hill.








Other Analogs of FM® 1-43A comparison of mammalian motor nerve terminals stained with either FM® 1-43 or the

more hydrophilic analog FM® 2-10 (T7508, Figure 16.1.22) revealed that lower background stain-ing by FM® 2-10 and its faster destaining rate may make it the preferred probe for quantitative applications.199,200 However, staining with FM® 2-10 requires much higher dye concentrations 199 (100 µM compared with 2 µM for FM® 1-43). Additionally, it has been shown that both FM® 1-43 and FM® 2-10 are antagonists of muscarinic acetylcholine receptors and may be useful for analyzing receptor distribution and occupancy.201 �is property may be due to the cationic alkylammonium substituent of FM® dyes, which they have in common with choline, and could serve as one of the sources of background FM® dye staining in tissues.

FM® 4-64 (T3166, T13320) and RH 414 (T1111)—both more hydrophobic than FM® 1-43—may also be useful as probes for investigating endocytosis. Because small di�erences in the polar-ity of these FM® probes can play a large role in their rates of uptake and their retention properties, we have introduced FM® 5-95 (T23360), a slightly less lipophilic analog of FM® 4-64 with es-sentially identical spectroscopic properties. FM® 4-64 exhibits long-wavelength red �uorescence that can be distinguished from Green Fluorescent Protein (GFP) with the proper optical �lter sets.202–205

FM® 4-64 is an endosomal marker and vital stain that persists through cell division,206,207 as well as a stain for functional presynaptic boutons.208 In addition, FM® 4-64 staining has been used to visualize membrane migration and fusion during Bacillus subtilis sporulation, and these movements can be correlated with the translocation of GFP-labeled proteins 202,209,210 (Figure 16.1.23). Sequential pulse-chase application of FM® 4-64 and FM® 1-43 allows two-color �uores-cence discrimination of temporally staged synaptic vesicle populations.187 FM® 4-64 selectively stains yeast vacuolar membranes and is an important tool for visualizing vacuolar organelle mor-phology and dynamics and for studying the endocytic pathway and vacuole fusion in yeast 211–213 (Section 12.3). FM® 4-64 and FM® 1-43 also have many applications for visualizing membrane dynamics in plant 204,214–216 and algal 217 cells.

FM® 1-43FX and FM® 4-64FX: Fixable FM® DyesFM® 1-43FX and FM® 4-64FX are FM® 1-43 and FM® 4-64 analogs, respectively, that have

been modi�ed to contain an aliphatic amine (Figure 16.1.24, Figure 16.1.25). �is modi�cation makes the probe �xable with aldehyde-based �xatives, including formaldehyde and glutaralde-hyde. FM® 1-43FX has been used to study synaptic vesicle cycling in cone photoreceptor termi-nals 187 and to investigate the functional maturation of glutamatergic synapses.218 FM® 1-43FX (F35355) and FM® 4-64FX (F34653) are available specially packaged in 10 vials of 100 µg each.

4-Di-1-ASP and 4-Di-2-ASP�e cationic mitochondrial dyes 4-Di-1-ASP (D288) and 4-Di-2-ASP (D289) stain presynap-

tic nerve terminals independent of neuronal activity.219–222 �ese aminostyrylpyridinium dyes have also been widely used as substrates for functional analysis of biogenic amine transport-ers 223–227 and renal and hepatic organic cation transporters.228–230

TMA-DPHAlso useful as a lipid marker for endocytosis and exocytosis is the cationic linear polyene

TMA-DPH (T204, Figure 16.1.26), which readily incorporates in the plasma membrane of live cells.231,232 TMA-DPH is virtually non�uorescent in water and is reported to bind to cells in pro-portion to the available membrane surface.233 Its �uorescence intensity is therefore sensitive to physiological processes that cause a net change in membrane surface area, making it an excellent probe for monitoring events such as changes in cell volume and exocytosis.233–236

Fluorescent Cholera Toxin Subunit B: Markers of Lipid RaftsFluorescent cholera toxins, which bind to galactosyl moieties, are markers of lipid ra�s—

regions of cell membranes high in ganglioside GM1 that are thought to be important in cell signaling.237,238 Lipid ra�s are detergent-insoluble, sphingolipid- and cholesterol-rich mem-brane microdomains that form lateral assemblies in the plasma membrane.239–245 Lipid ra�s also sequester glycophosphatidylinositol (GPI)-linked proteins and other signaling proteins and

Figure 16.1.23 Correlated �uorescence imaging of mem-brane migration, protein translocation and chromosome localization during Bacillus subtilis sporulation. Membranes were stained with red-�uorescent FM® 4-64 (T3166, T13320). Chromosomes were localized with the blue-�uorescent nu-clear counterstain DAPI (D1306, D3571, D21490). The small, green-�uorescent patches (top row) indicate the localization of a GFP fusion to SpoIIIE, a protein essential for both initial membrane fusion and forespore engulfment. Progression of the engulfment is shown from left to right. Green �uorescence in the middle and bottom rows demonstrates fully engulfed sporangia stained with MitoTracker® Green FM® (M7514). Full details of the experimental methods and interpretation are published in Proc Natl Acad Sci U S A 96, 14553 (1999). Image contributed by Kit Pogliano and Marc Sharp, University of California at San Diego. Reproduced from the 7 December 1999 issue of Proc Natl Acad Sci U S A, with permission.

Figure 16.1.24 FM® 1-43FX (F35355).

H��(CH2)� ��(CH2)�� CH CH ��(CH2)�CH��2

CH�

CH� ��C�

Figure 16.1.25 FM® 4-64FX (F34653).

(CH CH)� �(CH2CH�)2H��(CH2)��(CH2)��

CH�

CH

��C��C��

Figure 16.1.26 TMA-DPH (1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate)(T204).

Figure 16.1.22 Feline muscle spindle, a specialized sen-sory receptor unit that detects muscle length and changes in muscle length and velocity, was labeled with FM® 2-10 (T7508). Image contributed by Michael Chua, University of North Carolina at Chapel Hill.







receptors, which may be regulated by their selective interactions with these membrane microdo-mains.246–251 Recent research has demonstrated that lipid ra�s play a role in a variety of cellular processes—including the compartmentalization of cell-signaling events,252–259 the regulation of apoptosis 260–262 and the intracellular tra�cking of certain membrane proteins and lipids 263–265—as well as in the infectious cycles of several viruses and bacterial pathogens.266–271

�e Vybrant® Lipid Ra� Labeling Kits (V34403, V34404, V34405; Section 14.4) provide the key reagents for �uorescently labeling lipid ra�s in vivo with our bright and extremely pho-tostable Alexa Fluor® dyes (Figure 16.1.27, Figure 16.1.28). Live cells are �rst labeled with the green-�uorescent Alexa Fluor® 488, orange-�uorescent Alexa Fluor® 555 or red-�uorescent Alexa Fluor® 594 conjugate of cholera toxin subunit B (CT-B). �is CT-B conjugate binds to the pen-tasaccharide chain of plasma membrane ganglioside GM1, which selectively partitions into lipid ra�s.250,272,273 An antibody that speci�cally recognizes CT-B is then used to crosslink the CT-B–labeled lipid ra�s into distinct patches on the plasma membrane, which are easily visualized by �uorescence microscopy.274,275 Each Vybrant® Lipid Ra� Labeling Kit contains su�cient reagents to label 50 live-cell samples, including:

• Recombinant cholera toxin subunit B (CT-B) labeled with the Alexa Fluor® 488 (in Kit V34403), Alexa Fluor® 555 (in Kit V34404) or Alexa Fluor® 594 (in Kit V34405) dye

• Anti–cholera toxin subunit B antibody (anti–CT-B)• Concentrated phosphate-bu�ered saline (PBS)• Detailed labeling protocol

Cholera toxin subunit B and its conjugates are also established as superior tracers for retro-grade labeling of neurons.276,277 Cholera toxin subunit B conjugates bind to the pentasaccharide chain of ganglioside GM1 on neuronal cell surfaces and are actively taken up and transported; alternatively, they can be injected by iontophoresis. Unlike the carbocyanine-based neuronal tracers such as DiI (D282, D3911, V22885; Section 14.4), cholera toxin subunit B conjugates can be used on tissue sections that will be �xed and frozen.278

All of our cholera toxin subunit B conjugates are prepared from recombinant cholera toxin subunit B, which is completely free of the toxic subunit A, thus eliminating any concern for toxicity or ADP-ribosylating activity. �e Alexa Fluor® 488 (C22841, C34775), Alexa Fluor® 555 (C22843, C34776), Alexa Fluor® 594 (C22842, C34777) and Alexa Fluor® 647 (C34778) conju-gates of cholera toxin subunit B combine this versatile tracer with the superior brightness of our Alexa Fluor® dyes to provide sensitive and selective receptor labeling and neuronal tracing. We also o�er biotin-XX (C34779) and horseradish peroxidase (C34780) conjugates of cholera toxin subunit B for use in combination with diaminobenzidine (DAB) oxidation,279 tyramide signal ampli�cation (TSA) and Qdot® nanocrystal–streptavidin conjugates.280

Fluorescent Protein–Based Lipid Raft MarkersCellLight® plasma membrane expression vectors (C10606, C10607, C10608; Section 14.4)

generate cyan-, green- or red-auto�uorescent proteins fused to a plasma membrane targeting se-quence consisting of the 10 N-terminal amino acids of LcK tyrosine kinase (Lck10). �ese fusion proteins are lipid ra� markers with well established utility,90 providing alternatives to cholera toxin B conjugates or BODIPY® FL C5-ganglioside GM1

91 (B13950, B34401; Section 13.3) with the inherent advantages of long-lasting and titratable expression conferred by BacMam 2.0 vector technology (BacMam Gene Delivery and Expression Technology—Note 11.1).

Fluorescent Protein–Based Synaptic Vesicle MarkersCellLight® Synaptophysin-GFP (C10609) and CellLight® Synaptophysin-RFP (C10610) are

valuable counterparts to FM® dyes for visualizing the distribution and density of presynaptic sites in neurons both in vitro and in vivo. Synaptophysin is a synaptic vesicle membrane glycoprotein that is involved in the biogenesis and fusion of synaptic vesicles but is not essential for neurotrans-mitter release. It is found in virtually all synaptically active neurons in the brain and spinal cord. �ese CellLight® reagents incorporate all the customary advantages of BacMam 2.0 delivery tech-nology including high transduction e�ciency and long-lasting and titratable expression (BacMam Gene Delivery and Expression Technology—Note 11.1).

Figure 16.1.27 Live J774 macrophage cells labeled with BODIPY® FL C5-ganglioside GM1 and Alexa Fluor® 555 chol-era toxin subunit B conjugate. Live J774 macrophage cells labeled with BODIPY® FL C5-ganglioside GM1 (B13950) and then with Alexa Fluor® 555 cholera toxin subunit B conju-gate (C22843; also available as a component of V34404). Cells were then treated with anti–CT-B antibody (a compo-nent of V34404) to induce crosslinking. Yellow �uorescence indicates colocalization of the two dyes. Nuclei were stained with the blue-�uorescent Hoechst 33342 dye (H1399, H3570, H21492).

Figure 16.1.28 A J774 mouse macrophage cell stained with BODIPY® FL ganglioside GM1 (B13950) and Alexa Fluor® 555 dye–labeled cholera toxin subunit B. A J774 mouse macrophage cell sequentially stained with BODIPY® FL gan-glioside GM1 (B13950) and then with Alexa Fluor® 555 dye–labeled cholera toxin subunit B (C22843, C34776; also avail-able as a component of V34404). The cell was then treated with an anti–CT-B antibody (a component of V34404) to in-duce crosslinking. Alexa Fluor® 555 dye �uorescence (top panel, red) and BODIPY® FL dye �uorescence (middle panel, green) were imaged separately and overlaid to empha-size the coincident staining (bottom panel, yellow). Nuclei were stained with blue-�uorescent Hoechst 33258 (H1398, H3569, H21491).








Anti–Synapsin I AntibodySynapsin I is an actin-binding protein that is localized exclusively to synaptic vesicles and

thus serves as an reliable marker for synapses in brain and other neuronal tissues.283 Synapsin I inhibits neurotransmitter release, an e�ect that is abolished upon its phosphorylation by Ca2+/calmodulin–dependent protein kinase II 284 (CaM kinase II). Antibodies directed against synapsin I have proven valuable in molecular and neurobiology research, for example, to estimate synaptic density and to follow synaptogenesis.218,285

We o�er a rabbit polyclonal anti–bovine synapsin I antibody as an a�nity-puri�ed IgG frac-tion (A6442). �is antibody was isolated from rabbits immunized against bovine brain synapsin I but is also active against human, rat and mouse forms of the antigen; it has little or no activity against synapsin II. �e a�nity-puri�ed rabbit polyclonal antibody was fractionated from the serum using column chromatography in which bovine synapsin I was covalently bound to the column matrix. A�nity-puri�ed anti–synapsin I antibody is suitable for immunohistochemistry (Figure 16.1.29), western blots, enzyme-linked immunosorbent assays and immunoprecipitations. Our complete selection of antibodies can be found at www.invitrogen.com/handbook/antibodies.

High Molecular Weight Polar MarkersFluorescent Protein–Based Endosomal Markers

CellLight® Early Endosomes–GFP (O10104) and CellLight® Early Endosomes–RFP (O36231) provide BacMam expression vectors encoding fusions of GFP or RFP with the small GTPase Rab5a. Rab5a fusions with auto�uorescent proteins are sensitive and precise early endosome markers for real-time imaging of clathrin-mediated endocytosis in live cells.276,286,287 �ese CellLight® reagents incorporate all the customary advantages of BacMam 2.0 delivery technol-ogy, including high transduction e�ciency and long-lasting and titratable expression (BacMam Gene Delivery and Expression Technology—Note 11.1).

Figure 16.1.29 Peripheral neurons in mouse intestinal cryosections were labeled with rabbit anti–synapsin I an-tibody (A6442) and detected using Alexa Fluor® 488 goat anti–rabbit IgG antibody (A11008). This tissue was counter-stained with DAPI (D1306, D3571, D21490).

Figure 16.1.30 The pH sensitivity of pHrodo™ dextran. pHrodo™ 10,000 MW dextran (P10361) was reconsitituted in HEPES (20 mM)–bu�ered PBS and adjusted to pH values from pH 4 to pH 10. The intensity of �uorescence emission increas-es with increasing acidity, particularly in the pH 5–8 range.

550 650600 700

pH 10pH 8pH 7pH 6

pH 4pH 5

Wavelength (nm)

Fluo

resc

ence

em

issi

on

Ex = 540 nm

Figure 16.1.31 Time course of pHrodo™ E. coli BioParticles® (P35361) uptake by metastatic malignant melanoma cells. Cells were imaged at 37°C in the continued presence of 100 µg/mL pHrodo™ BioParticles®. Uptake of pHrodo™ BioParticles® was observable as early as 20 minutes and reached a plateau within 2 to 3 hours.

20 µm 20 µm 20 µm 20 µm

0 min 40 min 80 min 120 min

Table 16.3 BioParticles® �uorescent bacteria and yeast.

Label (Abs/Em Maxima in nm)

Escherichia coli (K-12 strain)

Staphylococcus aureus (Wood strain without protein A)

Zymosan A (Saccharomyces cerevisiae)

Fluorescein (494/518) E2861 S2851 Z2841

Alexa Fluor® 488 (495/519) E13231 S23371 Z23373

BODIPY® FL (505/513) E2864 S2854

Tetramethylrhodamine (555/580) E2862

pHrodo™ (560/585) A10025, P35361 A10010

Alexa Fluor® 594 (590/617) E23370 S23372 Z23374

Texas Red® (595/615) E2863 Z2843

Unlabeled S2859 Z2849

We also o�er opsonizing reagents for enhancing the uptake of BioParticles® products. These reagents are derived from puri�ed rabbit polyclonal IgG antibodies that are speci�c for the E. coli (E2870), S. aureus (S2860) or zymosan (Z2850) particles. Reconstitution of the lyophilized opsonizing reagents requires only the addition of water, and one unit of opsonizing reagent is su�cient to opsonize ~10 mg of the corresponding BioParticles® product.


IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofi sher.com/probes





BioParticles® Fluorescent Bacteria and Yeast�e BioParticles® product line consists of a series of �uorescently

labeled, heat- or chemically killed bacteria and yeast in a variety of sizes, shapes and natural antigenicities. �ese �uorescent BioParticles® products have been employed to study phagocytosis by �uorescence microscopy,288,289 quantitative spectro�uorometry 290 and �ow cytom-etry.288,291 We o�er Escherichia coli (K-12 strain), Staphylococcus aureus (Wood strain without protein A) and zymosan (Saccharomyces cerevi-siae) BioParticles® products covalently labeled with a variety of �uoro-phores, including Alexa Fluor®, �uorescein, BODIPY® FL, tetrameth-ylrhodamine, Texas Red® and pHrodo™ dyes (Table 16.3). Special care has been taken to remove any free dye a�er conjugation. BioParticles® products are freeze-dried and ready for reconstitution in a bu�er of choice and are supplied with a general protocol for measuring phagocy-tosis; we also o�er opsonizing reagents for use with each particle type, as described below.

Unlike the �uorescence of �uorescein-labeled BioParticles® bacteria and yeast, which is strongly quenched in acidic environments, the �uo-rescence of the Alexa Fluor® 488, BODIPY® FL, tetramethylrhodamine and Texas Red® BioParticles® conjugates is uniformly intense between pH 3 and 10. �is property is particularly useful for quantitating �uo-rescent bacteria and zymosan within acidic phagocytic vacuoles.

Fluorescent bacteria and yeast particles are proven tools for study-ing a variety of phagocytosis parameters. For example, they have been used to:

• Detect the phagocytosis of yeast by murine peritoneal macro-phage 292 and human neutrophils 290

• Determine the e�ects of di�erent opsonization procedures on the e�ciency of phagocytosis of pathogenic bacteria 293 and yeast 290

• Investigate the kinetics of phagocytosis degranulation and actin polymerization in stimulated leukocytes 290

• Quantitate the e�ects of purinergic P2X7 receptor activation on phagosomal maturation 179

• Show that Dictyostelium discoideum depleted of clathrin heavy chains are still able to undergo phagocytosis of �uorescent zymosans 294

• Study molecular defects in phagocytic function 178

Vybrant® Phagocytosis Assay Kit�e Vybrant® Phagocytosis Assay Kit (V6694) provides a conve-

nient set of reagents for quantitating phagocytosis and assessing the e�ects of certain drugs or conditions on this cellular process. In this assay, cells of interest are incubated �rst with green-�uorescent �uo-rescein-labeled E. coli BioParticles® conjugates, which are internalized by phagocytosis, and then with trypan blue, which quenches the �uo-rescence of any extracellular BioParticles® product (Figure 16.1.17). �e methodology provided by this kit was developed using the adherent murine macrophage cell line J774; 176 however, researchers have adapted this assay to other phagocytic cell types 295 and other instrument plat-forms such as �ow cytometers.296 Each kit provides su�cient reagents for 250 tests using a 96-well microplate format and contains:

• BioParticles® �uorescein-labeled Escherichia coli• Hanks’ balanced salt solution (HBSS)• Trypan blue• Step-by-step instructions for performing the phagocytosis assay

pHrodo™ BioParticles® Fluorescent BacteriaIn contrast to both the �uorescein- and Alexa Fluor® dye–labeled

BioParticles® conjugates, the �uorescence of the pHrodo™ E. coli and S. aureus BioParticles® conjugates (P35361, A10010) increases in acidic environments (Figure 16.1.30), providing a continuous positive indica-tor of phagocytic uptake. With a simple no-cell background subtraction method, a large and speci�c signal is obtained from cells that ingest the pHrodo™ BioParticles®, providing a speci�c index of phagocytosis in the context of a variety of pretreatments or conditions (Figure 16.1.31). �e optimal absorption and �uorescence emission maxima of the pHrodo™ BioParticles® conjugates are approximately 560 nm and 585 nm, respec-tively, but the pHrodo™ �uorophore is also readily excited by the 488 nm spectral line of the argon-ion laser used in most �ow cytometers.

With each pHrodo™ BioParticles® conjugate, we provide su�cient reagent for 100 microplate wells in a 96-well format, along with step-by-step instructions for performing phagocytosis assays in a �uorescence microplate reader. �is methodology has been developed using adher-ent J774A.1 murine macrophage cells, but can be adapted for use with other adherent cells,178 primary cells 179,297 or cells in suspension,298 as well as for in vivo applications.299 Cells assayed for phagocytic activ-ity with pHrodo™ BioParticles® conjugates may be �xed with standard formaldehyde solutions for later analysis, preserving di�erences in signal between control and experimental samples with high �delity. pHrodo™ BioParticles® conjugate preparations are also amenable to op-sonization (E2870, S2860), which can greatly enhance their uptake and signal strength in the phagocytosis assay.

To facilitate the use of pHrodo™ BioParticles® conjugates for the study of phagocytosis, we o�er the pHrodo™ E. coli BioParticles® Phagocytosis Kit for Flow Cytometry (A10025), which provides the key reagents for assessing particle ingestion and red blood cell lysis (Figure 16.1.32). Each kit provides su�cient reagents for performing 100 assays when using sample volumes of 100 µL whole blood per assay, including:

• pHrodo™ E. coli BioParticles® conjugates• Lysis and wash bu�ers• Detailed protocols

Figure 16.1.32 Flow cytometry analysis showing increased �uorescence of granulocytes treated with pHrodo™ E. coli BioParticles® (P35361). A whole blood sample was collected and treated with heparin, and two 100 µL aliquots were prepared. Both aliquots were treated with pHrodo™ BioParticles® and vortexed. One sample was placed in a 37°C water bath, and the other sample (negative control) was placed in an ice bath. After a 15-minute incubation, red blood cells were lysed with an ammonium chloride–based lysis bu�er. The samples were centrifuged for 5 minutes at 500 rcf, washed once, and resuspended with HBSS. The samples were then analyzed on a BD FACSCalibur™ cytometer (BD Biosciences) using a 488 nm argon laser and 564–606 nm emission �lter. A) Granulocytes were gated using forward and side scatter. B) The sample incubated at 37°C shows the increased �uorescence of the phagocy-tosed pHrodo™ BioParticles® (red), in contrast to the negative control sample, which was kept on ice to inhibit phagocytosis (blue).

Forward scatter

granulocytes

monocytes

lymphocytes

debris

0 800600400200 1,000

Sid

e sc

atte

r

200

400

600

800

1,000

0

pHrodo™ dye �uorescence (585 nm)

Num

ber

of c

ells

cou

nted

120

80

40

0100 101 102 103 104

negativecontrol

phagocytosedparticles

A B








pHrodo™ Phagocytosis Particle Labeling KitIn addition to the pHrodo™ BioParticles® conjugates, we o�er the pHrodo™ Phagocytosis

Particle Labeling Kit for Flow Cytometry (A10026), which allows rapid labeling of biological particles, such as bacteria, and subsequent assessment of of phagocytic activity in whole blood samples by �ow cytometry. Each kit provides su�cient reagents for performing 100 assays when using sample volumes of 100 µL whole blood per assay, including:

• pHrodo™ succinimidyl ester• Lysis and wash bu�ers• Dimethylsulfoxide (DMSO)• Sodium bicarbonate• Detailed protocols

�e amine-reactive pHrodo™ succinimidyl ester is also available separately (P36600, Section 20.4) for creating pH-sensitive conjugates for following phagocytosis. pHrodo™ succinimidyl es-ter was used to label dexamethasone-treated thymocytes for �ow cytometry detection of phago-cytosis by splenic or peritoneal macrophages.180

Opsonizing Reagents and Non�uorescent BioParticles® ProductsMany researchers may want to use autologous serum to opsonize their �uorescent zymosan

and bacterial particles; however, we also o�er special opsonizing reagents (E2870, S2860, Z2850) for enhancing the uptake of each type of particle, along with a protocol for opsonization. �ese reagents are derived from puri�ed rabbit polyclonal IgG antibodies that are speci�c for the E. coli, S. aureus or zymosan particles. Reconstitution of the lyophilized opsonizing reagents requires only the addition of water, and one unit of opsonizing reagent is su�cient to opsonize ~10 mg of the corresponding BioParticles® product.

In addition, we o�er non�uorescent zymosan (Z2849) and S. aureus (S2859) BioParticles® products. �ese non�uorescent BioParticles® products are useful either as controls or for custom labeling with the reactive dye or indicator of interest.

Fluorescent Polystyrene MicrospheresFluorescent polystyrene microspheres with diameters between 0.5 and 2.0 µm have been

used to investigate phagocytic processes in murine melanoma cells,300 human alveolar mac-rophages,289 ciliated protozoa 137 and Dictyostelium discoideum.301,302 �e phagocytosis of �uo-rescent microspheres has been quantitated both with image analysis 289,303,304 and with �ow cy-tometry.305 Section 6.5 includes a detailed description of our full line of FluoSpheres® (Table 6.7) and TransFluoSpheres® (Table 6.9) �uorescent microspheres. Because of their low nonspeci�c binding, carboxylate-modi�ed microspheres appear to be best for phagocytosis applications. For phagocytosis experiments involving multicolor detection, we particularly recommend our 1.0 µm TransFluoSpheres® �uorescent microspheres 306 (T8880, T8883; Section 6.5). Various op-sonizing reagents, such as rabbit serum or fetal calf serum, have been used with the microspheres to facilitate phagocytosis.

Fluorescent Microspheres Coated with CollagenFibroblasts phagocytose and subsequently digest collagen. �ese activities play an important

role in the remodeling of the extracellular matrix during normal physiological turnover of con-nective tissues and wound repair, as well as in development and aging. A well-established proce-dure for observing collagen phagocytosis by either �ow cytometry or �uorescence microscopy entails the use of collagen-coated �uorescent microspheres that attach to the cell surface and become engulfed by �broblasts.307 We o�er yellow-green–�uorescent FluoSpheres® collagen I–labeled microspheres in either 1.0 µm or 2.0 µm diameter (F20892, F20893) for use in these appli-cations. In the production of these microspheres, collagen I from calf skin is attached covalently to the microsphere’s surface.

Figure 16.1.33 Detection of endosomal fusion. A) Cells are �rst incubated with a combination of a high molecular weight, red-�uorescent dextran (D1829, D1830, D1864) and the green-�uorescent Oregon Green® 514 streptavidin (S6369), which intrinsically has low �uorescence. B) The cells are then incubated with a biotinylated probe, e.g., bio-tinylated transferrin (T23363), and the excess conjugate is washed. C) Endosomal fusion is monitored by an increase in �uorescence of the Oregon Green® 514 dye as it is displaced by the biotinylated protein. The red-�uorescent dextran’s �uorescence remains constant and allows for ratiometric measurements of the fused endosomes.

Dextran Oregon Green® 514streptavidin

Biotinylatedprotein

Endosomalfusion

A

B

C







Fluorescent DextransTracing internalization of extracellularly introduced �uorescent dextrans is a standard

method for analyzing �uid-phase endocytosis.2,73,308,309 We o�er dextrans with nominal mo-lecular weights ranging from 3000 to 2,000,000 daltons, many of which can also be used as pinocytosis or phagocytosis markers (see Section 14.5 and Table 14.4 for further discussion and a complete product list). Discrimination of internalized �uorescent dextrans from dextrans in the growth medium is facilitated by use of reagents that quench the �uorescence of the external probe. For example, most of our anti-�uorophore antibodies (Section 7.4, Table 7.8) strongly quench the �uorescence of the corresponding dyes.

Negative staining produced by �uorescent dextrans that have been intracellularly infused via a patch pipette is indicative of nonendocytic vacuoles in live pancreatic acinar cells.310 Extracellular addition of a second, color-contrasting dextran then allows discrimination of en-docytic and nonendocytic vacuoles. Intracellular fusion of endosomes has been monitored with a BODIPY® FL avidin conjugate by following the �uorescence enhancement that occurs when it complexes with a biotinylated dextran.311 We have found our Oregon Green® 514 streptavidin (S6369, Section 7.6) to have an over 15-fold increase in �uorescence intensity upon binding free biotin, which may make it the preferred probe for this application (Figure 16.1.33).

pH Indicator Dextrans�e �uorescein dextrans (pKa ~6.4) are frequently used to investigate endocytic acidi�ca-

tion.312,313 Fluorescence of �uorescein-labeled dextrans is strongly quenched upon acidi�cation; however, �uorescein’s lack of a spectral shi� in acidic solution makes it di�cult to discriminate between an internalized probe that is quenched and residual �uorescence of the external me-dium. Dextran conjugates that either shi� their emission spectra in acidic environments, such as the SNARF® dextrans (Section 20.4), or undergo signi�cant shi�s of their excitation spectra, such as BCECF and Oregon Green® dextrans (Section 20.4), provide alternatives to �uorescein. �e Oregon Green® 488 and Oregon Green® 514 dextrans exhibit a pKa of approximately 4.7, facilitating measurements in acidic environments.312,314 In addition to these pH indicator dex-trans, we prepare a dextran that is double-labeled with �uorescein and tetramethylrhodamine (D1951; Section 20.4), which has been used as a ratiometric indicator (Figure 16.1.34) to measure endosomal acidi�cation in Hep G2 cells 315 and murine alveolar macrophages.178

In contrast to �uorescein and Oregon Green® 488 dextrans, pHrodo™ 10,000 MW dextran (P10361) exhibits increasing �uorescence in response to acidi�cation 178 (Figure 16.1.30). �e minimal �uorescent signal from pHrodo™ dextran at neutral pH prevents the detection of nonin-ternalized and nonspeci�cally bound conjugates and eliminates the need for quenching reagents and extra wash steps, thus providing a simple �uorescent assay for endocytic activity. pHrodo™ dextran’s excitation and emission maxima of 560 and 585 nm, respectively, facilitate multiplex-ing with other �uorophores including blue-, green- and far-red–�uorescent probes. Although pHrodo™ dextran is optimally excited at approximately 560 nm, it is also readily excited by the 488 nm spectral line of the argon-ion laser found on �ow cytometers, confocal microscopes and imaging microplate readers (Figure 16.1.18).

Low Molecular Weight Polar MarkersHydrophilic �uorescent dyes—including sulforhodamine 101 (S359), lucifer yellow CH (L453),

calcein (C481), 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS, pyranine; H348) and Cascade Blue® hydrazide (C687)—are taken up by actively �ring neurons through endocytic recycling of the syn-aptic vesicles.316,317 Unlike the �uorescent FM® membrane probes described above, however, the hydrophilic �uorophores appear to work for only a limited number of species in this application. In some tissue preparations, background due to noninternalized polar markers is easier to wash away than that emanating from membrane markers such as FM® 1-43.316 �e same dyes have frequently been used as �uid-phase markers of pinocytosis.318–321 �e highly water-soluble Alexa Fluor® hydra-zides and Alexa Fluor® hydroxylamines (Section 14.3, Table 3.2) provide superior spectral proper-ties and can be �xed in cells by aldehyde-based �xatives.322

Figure 16.1.34 The excitation spectra of double-labeled �uorescein-tetramethylrhodamine dextran (D1951), which contains pH-dependent (�uorescein) and pH-independent (tetramethylrhodamine) dyes.

6.3

5.0

6.0

5.5

pH 8.07.06.6

Em = 580 nm

Fluo

resc

ence

exc

itatio

n

Wavelength (nm)450 500 550 600








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DATA TABLE 16.1 PROBES FOR FOLLOWING RECEPTOR BINDING AND PHAGOCYTOSISCat. No. MW Storage Soluble Abs EC Em Solvent NotesC481 622.54 L pH >5 494 77,000 517 pH 9 1C687 596.44 L H2O 399 30,000 421 H2O 2, 3D288 366.24 L DMF 475 45,000 605 MeOH 4D289 394.30 L H2O, DMF 488 48,000 607 MeOH 4D1383 840.98 L pH >6, DMF 494 76,000 519 pH 9D2935 584.37 F,D,AA DMF 258 11,000 none MeOH 5E3476 ~6100 FF,D H2O <300 noneE3477 ~6600 FF,D H2O <300 none 6E3478 ~6500 FF,D,L H2O 495 84,000 517 pH 8 6, 7E3480 see Notes FF,D,L H2O 596 ND 612 pH 7 8, 9E3481 ~6800 FF,D,L H2O 555 85,000 581 pH 7 6, 7E7498 ~6600 FF,D,L H2O 511 85,000 528 pH 9 6, 7E13345 see Notes FF,D,L H2O 497 ND 520 pH 8 8, 10E35350 see Notes FF,D,L H2O 554 ND 568 pH 7 8, 11E35351 see Notes FF,D,L H2O 653 ND 671 pH 7 8, 12F1314 1213.41 F,L pH >6, DMF 494 72,000 517 pH 9F2902 see Notes RR,L,AA H2O <300 none 13, 14, 15F34653 788.75 D,L H2O, DMSO 562 47,000 744 CHCl3 4F35355 560.09 D,L H2O, DMSO 510 50,000 626 MeOH 4H348 524.37 D,L H2O 454 24,000 511 pH 9 16L453 457.24 L H2O 428 12,000 536 H2O 17, 18L3482 see Notes RR,L,AA see Notes 554 ND 571 see Notes 8, 19, 20, 21L3483 see Notes RR,L,AA see Notes 515 ND 520 see Notes 8, 19, 20, 21L3484 see Notes RR,L,AA see Notes 554 ND 571 see Notes 8, 19, 20, 21L3485 see Notes RR,L,AA see Notes 510 ND 518 see Notes 8, 19, 20, 21L23380 see Notes RR,L,AA see Notes 495 ND 519 see Notes 8, 19, 20, 21S359 606.71 L H2O 586 108,000 605 H2OT204 461.62 D,L DMF, DMSO 355 75,000 430 MeOH 22T1111 581.48 D,L DMSO, EtOH 532 55,000 716 MeOH 4, 23T3163 611.55 D,L H2O, DMSO 471 38,000 581 see Notes 24, 25T3166 607.51 D,L H2O, DMSO 505 47,000 725 see Notes 24, 26T7508 555.44 D,L H2O, DMSO 506 50,000 620 MeOH 4T13320 607.51 D,L H2O, DMSO 505 47,000 725 see Notes 24, 26T23360 565.43 D,L H2O, DMSO 560 43,000 734 CHCl3 26T35356 611.55 D,L H2O, DMSO 471 38,000 581 see Notes 24, 25For de�nitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.Notes

1. C481 �uorescence is strongly quenched by micromolar concentrations of Fe3+, Co2+, Ni2+ and Cu2+ at pH 7. (Am J Physiol (1995) 268:C1354, J Biol Chem (1999) 274:13375)2. The Alexa Fluor® 405 and Cascade Blue® dyes have a second absorption peak at about 376 nm with EC ~80% of the 395–400 nm peak.3. Maximum solubility in water is ~1% for C687, ~1% for C3221 and ~8% for C3239.4. Abs and Em of styryl dyes are at shorter wavelengths in membrane environments than in reference solvents such as methanol. The di�erence is typically 20 nm for absorption and 80 nm for

emission, but varies considerably from one dye to another. Styryl dyes are generally non�uorescent in water.5. Dihydro�uorescein diacetates are colorless and non�uorescent until both of the acetate groups are hydrolyzed and the products are subsequently oxidized to �uorescein derivatives. The materi-

als contain less than 0.1% of oxidized derivative when initially prepared. The oxidation products of C400, C2938, C6827, D399 and D2935 are 2’,7’-dichloro�uorescein derivatives with spectra similar to C368 (see data).

6. α-Bungarotoxin, EGF and phallotoxin conjugates have approximately 1 label per peptide.7. The value of EC listed for this EGF conjugate is for the labeling dye in free solution. Use of this value for the conjugate assumes a 1:1 dye:peptide labeling ratio and no change of EC due to dye–

peptide interactions.8. ND = not determined.9. E3480 is a complex of E3477 with Texas Red® streptavidin, which typically incorporates 3 dyes/streptavidin (MW ~52,800).10. E13345 is a complex of E3477 with Alexa Fluor® 488 streptavidin, which typically incorporates 5 dyes/streptavidin (MW ~52,800).11. E35350 is a complex of E3477 with Alexa Fluor® 555 streptavidin, which typically incorporates 3 dyes/streptavidin (MW ~52,800).12. E35351 is a complex of E3477 with Alexa Fluor® 647 streptavidin, which typically incorporates 3 dyes/streptavidin (MW ~52,800).13. This product is supplied as a ready-made solution in the solvent indicated under “Soluble.”14. F2902 is essentially colorless and non�uorescent until oxidized. A small amount (~5%) of oxidized material is normal and acceptable for the product as supplied. The oxidation product is

�uorescent (Abs = 495 nm, Em = 524 nm). (J Immunol Methods (1990) 130:223)15. This product consists of a dye–bovine serum albumin conjugate (MW ~66,000) complexed with IgG in a ratio of approximately 1:4 mol:mol (BSA:IgG)16. H348 spectra are pH-dependent.17. The �uorescence quantum yield of lucifer yellow CH in H2O is 0.21. (J Am Chem Soc (1981) 103:7615)18. Maximum solubility in water is ~8% for L453, ~6% for L682 and ~1% for L1177.19. LDL complexes must be stored refrigerated BUT NOT FROZEN. The maximum shelf-life under the indicated storage conditions is 4–6 weeks.20. This LDL complex incorporates multiple �uorescent labels. The number of dyes per apoprotein B (MW ~500,000) is indicated on the product label.21. LDL complexes are packaged under argon in 10 mM Tris, 150 mM NaCl, 0.3 mM EDTA, pH 8.3 containing 2 mM azide. Spectral data reported are measured in this bu�er.22. Diphenylhexatriene (DPH) and its derivatives are essentially non�uorescent in water. Absorption and emission spectra have multiple peaks. The wavelength, resolution and relative intensity of

these peaks are environment dependent. Abs and Em values are for the most intense peak in the solvent speci�ed.23. RH 414 Abs ~500 nm, Em ~635 nm when bound to phospholipid bilayer membranes.24. Abs, EC and Em determined for dye bound to detergent micelles (20 mg/mL CHAPS in H2O). These dyes are essentially non�uorescent in pure water.25. FM® 1-43 Abs = 479 nm, Em = 598 nm bound to phospholipid bilayer membranes. Em = 565 nm bound to synaptosomal membranes. (Neuron (1994) 12:1235)26. FM® 4-64 and FM® 5-95 are non�uorescent in water. For two-color imaging in GFP-expressing cells, these dyes can be excited at 568 nm with emission detection at 690–730 nm. (Am J Physiol

Cell Physiol (2001) 281:C624)








PRODUCT LIST 16.1 PROBES FOR FOLLOWING RECEPTOR BINDING AND PHAGOCYTOSISCat. No. Product QuantityA6442 anti-synapsin I (bovine), rabbit IgG fraction *a�nity puri�ed* 10 µgA11130 anti-transferrin receptor (human), mouse IgG1, monoclonal 236-15375 50 µgC481 calcein *high purity* 100 mgC687 Cascade Blue® hydrazide, trisodium salt 10 mgC2990 casein, �uorescein conjugate 25 mgC10586 CellLight® Early Endosomes-GFP 1 kitC10587 CellLight® Early Endosomes-RFP 1 kitC10609 CellLight® Synaptophysin-GFP 1 kitC10610 CellLight® Synaptophysin-RFP 1 kitC34775 cholera toxin subunit B (recombinant), Alexa Fluor® 488 conjugate 100 µgC22841 cholera toxin subunit B (recombinant), Alexa Fluor® 488 conjugate 500 µgC34776 cholera toxin subunit B (recombinant), Alexa Fluor® 555 conjugate 100 µgC22843 cholera toxin subunit B (recombinant), Alexa Fluor® 555 conjugate 500 µgC34777 cholera toxin subunit B (recombinant), Alexa Fluor® 594 conjugate 100 µgC22842 cholera toxin subunit B (recombinant), Alexa Fluor® 594 conjugate 500 µgC34778 cholera toxin subunit B (recombinant), Alexa Fluor® 647 conjugate 100 µgC34779 cholera toxin subunit B (recombinant), biotin-XX conjugate 100 µgC34780 cholera toxin subunit B (recombinant), horseradish peroxidase conjugate 100 µgD1383 dexamethasone �uorescein 5 mgD2935 2’,7’-dichlorodihydro�uorescein diacetate, succinimidyl ester (OxyBURST® Green H2DCFDA, SE) 5 mgD289 4-(4-(diethylamino)styryl)-N-methylpyridinium iodide (4-Di-2-ASP) 1 gD288 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (4-Di-1-ASP) 1 gD12060 DQ™ collagen, type I from bovine skin, �uorescein conjugate 1 mgD12054 DQ™ gelatin from pig skin, �uorescein conjugate *special packaging* 5 x 1 mgD12050 DQ™ Green BSA *special packaging* 5 x 1 mgD12053 DQ™ ovalbumin *special packaging* 5 x 1 mgD12051 DQ™ Red BSA *special packaging* 5 x 1 mgE3476 epidermal growth factor (EGF) *from mouse submaxillary glands* 100 µgE3477 epidermal growth factor, biotin-XX conjugate (biotin EGF) 20 µgE13345 epidermal growth factor, biotinylated, complexed to Alexa Fluor® 488 streptavidin (Alexa Fluor® 488 EGF complex) 100 µgE35350 epidermal growth factor, biotinylated, complexed to Alexa Fluor® 555 streptavidin (Alexa Fluor® 555 EGF complex) 100 µgE35351 epidermal growth factor, biotinylated, complexed to Alexa Fluor® 647 streptavidin (Alexa Fluor® 647 EGF complex) 100 µgE3480 epidermal growth factor, biotinylated, complexed to Texas Red® streptavidin (Texas Red® EGF complex) 100 µgE3478 epidermal growth factor, �uorescein conjugate (�uorescein EGF) 20 µgE7498 epidermal growth factor, Oregon Green® 514 conjugate (Oregon Green® 514 EGF) 20 µgE3481 epidermal growth factor, tetramethylrhodamine conjugate (rhodamine EGF) 20 µgE2870 Escherichia coli BioParticles® opsonizing reagent 1 UE13231 Escherichia coli (K-12 strain) BioParticles®, Alexa Fluor® 488 conjugate 2 mgE23370 Escherichia coli (K-12 strain) BioParticles®, Alexa Fluor® 594 conjugate 2 mgE2864 Escherichia coli (K-12 strain) BioParticles®, BODIPY® FL conjugate 10 mgE2861 Escherichia coli (K-12 strain) BioParticles®, �uorescein conjugate 10 mgE2862 Escherichia coli (K-12 strain) BioParticles®, tetramethylrhodamine conjugate 10 mgE2863 Escherichia coli (K-12 strain) BioParticles®, Texas Red® conjugate 10 mgF2902 Fc OxyBURST® Green assay reagent *25 assays* *3 mg/mL* 500 µLF13191 �brinogen from human plasma, Alexa Fluor® 488 conjugate 5 mgF13192 �brinogen from human plasma, Alexa Fluor® 546 conjugate 5 mgF13193 �brinogen from human plasma, Alexa Fluor® 594 conjugate 5 mgF35200 �brinogen from human plasma, Alexa Fluor® 647 conjugate 5 mgF7496 �brinogen from human plasma, Oregon Green® 488 conjugate 5 mgF20892 FluoSpheres® collagen I-labeled microspheres, 1.0 µm, yellow-green �uorescent (505/515) *0.5% solids* 0.4 mLF20893 FluoSpheres® collagen I-labeled microspheres, 2.0 µm, yellow-green �uorescent (505/515) *0.5% solids* 0.4 mLF35355 FM® 1-43FX *�xable analog of FM® 1-43 membrane stain* 10 x 100 µgF34653 FM® 4-64FX *�xable analog of FM® 4-64 membrane stain* 10 x 100 µgF1314 formyl-Nle-Leu-Phe-Nle-Tyr-Lys, �uorescein derivative 1 mgG13187 gelatin from pig skin, �uorescein conjugate 5 mgG13186 gelatin from pig skin, Oregon Green® 488 conjugate 5 mgH13188 histone H1 from calf thymus, Alexa Fluor® 488 conjugate 1 mgH348 8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt (HPTS; pyranine) 1 gI13269 insulin, human, recombinant from E. coli, �uorescein conjugate (FITC insulin) *monolabeled* *zinc free* 100 µg







PRODUCT LIST 16.1 PROBES FOR FOLLOWING RECEPTOR BINDING AND PHAGOCYTOSIS—continuedCat. No. Product QuantityL21409 lectin PNA from Arachis hypogaea (peanut), Alexa Fluor® 488 conjugate 1 mgL32458 lectin PNA from Arachis hypogaea (peanut), Alexa Fluor® 568 conjugate 1 mgL32459 lectin PNA from Arachis hypogaea (peanut), Alexa Fluor® 594 conjugate 1 mgL32460 lectin PNA from Arachis hypogaea (peanut), Alexa Fluor® 647 conjugate 1 mgL23351 lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 488 conjugate 100 µgL23352 lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 568 conjugate 100 µgL23353 lipopolysaccharides from Escherichia coli serotype 055:B5, Alexa Fluor® 594 conjugate 100 µgL23350 lipopolysaccharides from Escherichia coli serotype 055:B5, BODIPY® FL conjugate 100 µgL23356 lipopolysaccharides from Salmonella minnesota, Alexa Fluor® 488 conjugate 100 µgL3486 low-density lipoprotein from human plasma (LDL) *2.5 mg/mL* 200 µLL35354 low-density lipoprotein from human plasma, acetylated (AcLDL) *2.5 mg/mL* 200 µLL23380 low-density lipoprotein from human plasma, acetylated, Alexa Fluor® 488 conjugate (Alexa Fluor® 488 AcLDL) *1 mg/mL* 200 µLL35353 low-density lipoprotein from human plasma, acetylated, Alexa Fluor® 594 conjugate (Alexa Fluor® 594 AcLDL) *1 mg/mL* 200 µLL3485 low-density lipoprotein from human plasma, acetylated, BODIPY® FL conjugate (BODIPY® FL AcLDL) *1 mg/mL* 200 µLL3484 low-density lipoprotein from human plasma, acetylated, DiI complex (DiI AcLDL) *1 mg/mL* 200 µLL3483 low-density lipoprotein from human plasma, BODIPY® FL complex (BODIPY® FL LDL) *1 mg/mL* 200 µLL3482 low-density lipoprotein from human plasma, DiI complex (DiI LDL) *1 mg/mL* 200 µLL453 lucifer yellow CH, lithium salt 25 mgO13291 OxyBURST® Green H2HFF BSA *special packaging* 5 x 1 mgP10361 pHrodo™ dextran, 10,000 MW *for endocytosis* 0.5 mgP35361 pHrodo™ E. coli BioParticles® conjugate for phagocytosis 5 x 2 mgA10025 pHrodo™ E. coli BioParticles® Phagocytosis Kit *for �ow cytometry* *100 tests* 1 kitA10026 pHrodo™ Phagocytosis Particle Labeling Kit *for �ow cytometry* *100 tests* 1 kitA10010 pHrodo™ S. aureus BioParticles® conjugate for phagocytosis 5 x 2 mgS2860 Staphylococcus aureus BioParticles® opsonizing reagent 1 US23371 Staphylococcus aureus (Wood strain without protein A) BioParticles®, Alexa Fluor® 488 conjugate 2 mgS23372 Staphylococcus aureus (Wood strain without protein A) BioParticles®, Alexa Fluor® 594 conjugate 2 mgS2854 Staphylococcus aureus (Wood strain without protein A) BioParticles®, BODIPY® FL conjugate 10 mgS2851 Staphylococcus aureus (Wood strain without protein A) BioParticles®, �uorescein conjugate 10 mgS2859 Staphylococcus aureus (Wood strain without protein A) BioParticles®, unlabeled 100 mgS359 sulforhodamine 101 25 mgT204 TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate) 25 mgT13342 transferrin from human serum, Alexa Fluor® 488 conjugate 5 mgT23364 transferrin from human serum, Alexa Fluor® 546 conjugate 5 mgT35352 transferrin from human serum, Alexa Fluor® 555 conjugate 5 mgT23365 transferrin from human serum, Alexa Fluor® 568 conjugate 5 mgT13343 transferrin from human serum, Alexa Fluor® 594 conjugate 5 mgT23362 transferrin from human serum, Alexa Fluor® 633 conjugate 5 mgT23366 transferrin from human serum, Alexa Fluor® 647 conjugate 5 mgT35357 transferrin from human serum, Alexa Fluor® 680 conjugate 5 mgT23363 transferrin from human serum, biotin-XX conjugate 5 mgT2871 transferrin from human serum, �uorescein conjugate 5 mgT2872 transferrin from human serum, tetramethylrhodamine conjugate 5 mgT2875 transferrin from human serum, Texas Red® conjugate 5 mgT3163 N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM® 1-43) 1 mgT35356 N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide (FM® 1-43) *special packaging* 10 x 100 µgT1111 N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)pyridinium dibromide (RH 414) 5 mgT3166 N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM® 4-64) 1 mgT13320 N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM® 4-64) *special packaging* 10 x 100 µgT7508 N-(3-triethylammoniumpropyl)-4-(4-(diethylamino)styryl)pyridinium dibromide (FM® 2-10) 5 mgT23360 N-(3-trimethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM® 5-95) 1 mgT23011 trypsin inhibitor from soybean, Alexa Fluor® 488 conjugate 1 mgV6694 Vybrant® Phagocytosis Assay Kit *250 assays* 1 kitZ2850 zymosan A BioParticles® opsonizing reagent 1 UZ23373 zymosan A (S. cerevisiae) BioParticles®, Alexa Fluor® 488 conjugate 2 mgZ23374 zymosan A (S. cerevisiae) BioParticles®, Alexa Fluor® 594 conjugate 2 mgZ2841 zymosan A (S. cerevisiae) BioParticles®, �uorescein conjugate 10 mgZ2843 zymosan A (S. cerevisiae) BioParticles®, Texas Red® conjugate 10 mgZ2849 zymosan A (S. cerevisiae) BioParticles®, unlabeled 100 mg







Section 16.2 Probes for Neurotransmitter Receptors

16.2 Probes for Neurotransmitter ReceptorsFluorescent receptor ligands provide a sensitive means of identi-

fying and localizing various cellular receptors, ion channels and ion carriers. Many of these site-selective �uorescent probes may be used on live or �xed cells, as well as in cell-free extracts. �e high sensitivity and selectivity of these �uorescent probes make them especially good candidates for measuring low-abundance receptors.1–5 Various meth-ods for further amplifying detection of these receptors 6,7 are discussed in Chapter 6 and Chapter 7.

�is section is devoted to our probes for neurotransmitter recep-tors. Additional �uorescently labeled receptor ligands (including low-density lipoproteins, epidermal growth factors, transferrin and �brino-gen conjugates and chemotactic peptides) are described in Section 16.1, along with other probes for studying receptor-mediated endocytosis. Section 16.3 describes a variety of probes for Ca2+, Na+, K+ and Cl– ion channels and carriers. Chapter 17 focuses on reagents for investigating events—such as calcium regulation, kinase, phosphatase and phospho-lipase activation, and lipid tra�cking—that occur downstream from the receptor–ligand interaction (Figure 16.2.1).

α-Bungarotoxin Probes for Nicotinic Acetylcholine ReceptorsFluorescent α-Bungarotoxins

Nicotinic acetylcholine receptors (nAChRs) are neurotransmitter-gated ion channels that produce an increase in Na+ and K+ permeabil-ity, depolarization and excitation upon activation by acetylcholine8 (Figure 16.2.1). α-Bungarotoxin is a 74–amino acid (~8000 dalton) peptide containing 5 lysine residues and 10 cysteine residues paired in 5 disul�de bridges. Extracted from Bungarus multicinctus venom, α-bungarotoxin binds with high a�nity to the α-subunit of the nAChR of neuromuscular junctions.9 We provide an extensive selection of �uorescent α-bungarotoxin conjugates (Table 16.4) to facilitate visu-alization of nAChRs with a variety of instrumentation. We attach ap-proximately one �uorophore to each molecule of α-bungarotoxin, thus retaining optimal binding speci�city. �e labeled bungarotoxins are then chromatographically separated from unlabeled molecules to en-sure adequate labeling of the product.

Alexa Fluor® 488 α-bungarotoxin (B13422) has �uorescence spectra similar to those of �uorescein α-bungarotoxin (F1176) and is

therefore suitable for use with standard �uorescein optical �lter sets. Tetramethylrhodamine α-bungarotoxin 10–12 (T1175) has been the pre-ferred red-orange–�uorescent probe for staining the nAChR (Figure 16.2.2). We not only o�er the red-orange–�uorescent Alexa Fluor® 555 α-bungarotoxin (B35451), but also the red-�uorescent Alexa Fluor® 594 α-bungarotoxin (B13423), which has a longer-wavelength emis-sion maximum and therefore o�ers better spectral separation from green-�uorescent dyes in multicolor experiments. Our two longest-wavelength conjugates—Alexa Fluor® 647 α-bungarotoxin (B35450) and Alexa Fluor® 680 α-bungarotoxin (B35452)—are spectrally sepa-rated from both green-�uorescent and orange-�uorescent dyes, allow-ing researchers to easily perform three- and four-color experiments.

Fluorescent α-bungarotoxins have been used in a variety of infor-mative investigations to:

• Correlate receptor clustering during neuromuscular development with tyrosine phosphorylation of the receptor 13,14

• Detect reinnervation of adult muscle a�er nerve damage and to identify and visualize endplates 15,16

• Document nAChR cluster formation a�er myoblast fusion.17

• Label proteins fused to the BBS expression tag (a 13–amino acid sequence excerpted from the nAChR) in situ 18,19

• Monitor nAChR-mediated responses in neuromuscular damage and degeneration models 20–22

Biotinylated α-BungarotoxinNicotinic AChRs can also be labeled with biotinylated

α-bungarotoxin (B1196), which is then localized using �uorophore- or enzyme-labeled avidin, streptavidin or NeutrAvidin biotin-binding protein conjugates, or NANOGOLD and Alexa Fluor® FluoroNanogold streptavidin 14,23–25 (Section 7.6, Table 7.9). Based on the intracellular dissociation of biotinylated α-bungarotoxin and streptavidin, re-searchers were able to distinguish new, preexisting and recycled pools of nAChR at the synapses of live mice by sequentially labeling with biotinylated α-bungarotoxin and �uorescent streptavidin conjugates.26 Complexation of biotinylated α-bungarotoxin with Qdot® nanocrys-tal–streptavidin conjugates (Section 6.6) enables single-molecule de-tection of nAChR.1,2 �e nanocrystal labeling methodology allows detection and tracking of di�use, nonclustered nAChRs, whereas dye-labeled α-bungarotoxin conjugates primarily detect nAChR clusters.1

Table 16.4 Labeled and unlabeled α-bungarotoxins.

Cat. No. Label Ex/Em (nm) Notes Size

F1176 Fluorescein 494/518 Original green-�uorescent conjugate 500 µg

B13422 Alexa Fluor® 488 495/519 Brightest and most photostable green-�uorescent conjugate 500 µg

T1175 Tetramethylrhodamine 553/577 An extensively used red-orange–�uorescent conjugate 500 µg

B35451 Alexa Fluor® 555 555/565 Bright and photostable red-orange–�uorescent conjugate 500 µg

B13423 Alexa Fluor® 594 590/617 Excellent dye to combine with green-�uorescent probes 500 µg

B35450 Alexa Fluor® 647 650/668 Excellent dye to combine with green- and orange-�uorescent probes 500 µg

B35452 Alexa Fluor® 680 679/702 Excellent dye to combine with green-, orange-, and red-�uorescent probes 500 µg

B1196 Biotin-XX NA Visualized with labeled avidins and streptavidins (Table 7.9) 500 µg

B1601 Unlabeled NA Useful as a control, as well as for radioiodination and for preparation of new conjugates 1 mg

NA = Not applicable.







Figure 16.2.1 Neurotransmitter receptors linked to second messengers mediating growth responses in neuronal and nonneuronal cells. Abbreviations: RAC/Gs = Receptors coupled to G-proteins that stimulate adenylate cyclase (AC) activity, leading to cAMP formation and enhanced activity of protein kinase A (PKA). RAC/Gi = Receptors coupled to pertussis toxin (PTX)–sensi-tive G-proteins that inhibit adenylate cyclase activity. RPLC = Receptors promoting the hydrolysis of phosphatidylinositol 4,5-diphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3), which increas-es intracellular Ca2+, and diacylglycerol (DAG), which activates protein kinase C (PKC). RION = Receptors indirectly promoting ion �uxes due to coupling to various G-proteins. RLG/ION = Receptors that promote ion �uxes directly because they are structurally linked to ion channels (members of the superfamily of ligand-gated ion channel receptors). Stimulation of proliferation is most often associated with activation of G-proteins negatively coupled to adenylate cyclase (Gi), or positively coupled to phospholipase C (Gq) or to pertussis toxin–sensitive pathways (Go, Gi). In con-trast, activation of neurotransmitter receptors positively coupled to cAMP usually inhibits cell proliferation and causes changes in cell shape indicative of di�erentiation. Reprinted and modi�ed with permission from J.M. Lauder and Trends Neurosci (1993) 16:233.

Ion channel–linkedG-protein–coupled receptors

Phospholipase C–linkedG-protein–coupled receptors

Adenylate cyclase–linkedG-protein–coupled receptors

5-HT3 (Na+, K+)Nicotinic (Na+)GABAA (Cl–)Ionotropic glutamate (Ca2+, Na+)

5-HT1A5-HT4

β-AdrenergicD4 Dopaminergic

A2 AdenosineVIP

Calcium can in�uence cellproliferation, neurite elongation,gene expression and cell viability.

5-HT1A (K+)5-HT1C (Cl–)5-HT2 (Cl–)β-Adrenergic (Ca2+, Na+)α2-Adrenergic (Ca2+, K+)Muscarinic (K+, Ca2+)D2 Dopaminergic (Ca2+, K+)GABAB (K+)

5-HT1A (transfected cells)5-HT1C (transfected cells)5-HT2 (transfected cells)α1-AdrenergicMuscarinicMetabotropic glutamate

Gs

Gi

RAC

PTX

+

Na+

K+

Cl–

Ca2+

–

PTX

IonchannelsGs

Gi

GqACGs GoPLC

5-HT1A5-HT1B5-HT1Dαt-AdrenergicMuscarinicD2 DopaminergicA1 AdenosineOpioidGABAB

RIONRPLC

Gi

ATP

PKA

cAMP+Pi Na+

K+

Cl–

Ca2+

Ligand-gated ion channels

Cell proliferation.

Cell proliferation.

RAC/Gs

RAC/Gi

RPLC

RION

RLG/ION

Gene expression (cAMP response elements), protein phosphorylation,changes in process outgrowth, secretion of growth factors from glia.

Neurite elongation.

RLG/ION

DAG +

PKC Ca2+

IP3PIP2

( )

( )

( )

( ) ( ) ( )

( )

Figure 16.2.2 Pseudocolored photomicrograph of the synaptic region of �uorescently labeled living muscle �bers from the lumbricalis muscle of the adult frog Rana pipiens. Six hours after isolation of the muscle �bers, acetylcholine receptors were stained with red-�uorescent tetramethyl-rhodamine α-bungarotoxin (T1175) and myonuclei were stained with the green-�uorescent SYTO® 13 live-cell nucleic acid stain (S7575). Photo contributed by Christian Brösamle, Brain Research Institute, University of Zurich, and Damien Ku�er, Institute of Neurobiology, University of Puerto Rico.

In addition, the biotinylated toxin can be employed for a�nity isolation of the nAChR using a streptavidin or CaptAvidin™ agarose (S951, C21386; Section 7.6) column.27,28

Unlabeled α-BungarotoxinIn addition to the �uorescent and biotinylated derivatives, we have unlabeled α-bungarotoxin

(B1601), which has been shown to be useful for radioiodination.9,29 Unlabeled α-bungarotoxin has also been employed for ELISA testing of nAChR binding,30 as well as for investigating the function of the α-bungarotoxin–binding component (α-BgtBC) in vertebrate neurons.31

Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit�e action of acetylcholine (ACh) at neuromuscular junctions is regulated by acetylcho-

linesterase (AChE), the enzyme that hydrolyzes ACh to choline and acetate. �e Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217) provides an ultrasensitive method for continuously monitoring AChE activity and for detecting ACh in a �uorescence microplate reader or �uorometer. Other potential uses for this kit include screening for AChE inhibi-tors and measuring the release of ACh from synaptosomes. �e Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit can also be used for the ultrasensitive, speci�c assay of free choline, classi�ed as an essential nutrient in foods.32








In this assay, AChE activity is monitored indirectly using the Amplex® Red reagent (10-ace-tyl-3,7-dihydroxyphenoxazine), a highly sensitive and stable �uorogenic probe for H2O2 that is also useful in assaying other enzymes and analytes (Section 10.5). First, AChE converts the acetylcholine substrate to choline. Choline is in turn oxidized by choline oxidase to betaine and H2O2, the latter of which, in the presence of horseradish peroxidase, reacts with the Amplex® Red reagent to generate the red-�uorescent product resoru�n (R363, Section 10.1) with excitation/emission maxima of ~570/585 nm (Figure 16.2.3). Experiments with puri�ed AChE from electric eel indicate that the Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit can detect AChE levels as low as 0.002 U/mL using a reaction time of only 1 hour (Figure 16.2.4). In our labora-tories, we have been able to detect acetylcholinesterase activity from a tissue sample with total protein content as low as 200 ng/mL or 20 ng/well in a microplate assay.33 By providing an excess of AChE in the assay, the kit can also be used to detect acetylcholine levels as low as 0.3 µM, with a detection range between 0.3 µM and ~100 µM acetylcholine (Figure 16.2.5).

�e Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit contains:

• Amplex® Red reagent• Dimethylsulfoxide (DMSO)• Horseradish peroxidase (HRP)• H2O2 for use as a positive control• Concentrated reaction bu�er• Choline oxidase from Alcaligenes sp.• Acetylcholine (ACh)• Acetylcholinesterase (AChE) from electric eel• Detailed protocols

Each kit provides su�cient reagents for approximately 500 assays using a �uorescence mi-croplate reader and a reaction volume of 200 µL per assay.

BODIPY® FL Prazosin for α1-Adrenergic ReceptorsPrazosin is a high-a�nity antagonist for the α1-adrenergic receptor. �e green-�uorescent

BODIPY® FL prazosin (B7433, Figure 16.2.6) can be used to localize the α1-adrenergic receptor on cultured cortical neurons 34 and in vascular smooth muscle cells from α1-adrenergic recep-tor–knockout mice.35 BODIPY® FL prazosin has also been successfully employed in multidrug resistance (MDR) transporter activity assays.36,37

BODIPY® TMR-X Muscimol for GABAA ReceptorsMuscimol is a powerful agonist of the GABAA receptor and has been widely used to revers-

ibly inactivate localized groups of neurons.38,39 Using red-�uorescent BODIPY® TMR-X mus-cimol (M23400, Figure 16.2.7), researchers can correlate the distribution of muscimol with its pharmacological e�ects 40 and detect the presence of GABAA receptors on cell surfaces.41

Fluorescent Angiotensin II for AT1 and AT2 ReceptorsAngiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) stimulates smooth muscle contraction

and plays an important role in blood pressure control and in water and salt homeostasis. �ese e�ects are exerted via two G-protein–coupled receptor subtypes, referred to as AT1 and AT2. Our N-terminal–labeled �uorescein and Alexa Fluor® 488 analogs of angiotensin II (A13438, A13439) are useful tools for imaging the distribution of these receptors,42,43 as well as for �ow cytometric analysis of angiotensin II endocytosis.44 �ese �uorescent peptides have been char-acterized for purity by HPLC and mass spectrometry and generally display selectivity for AT1 over AT2 binding.42

Figure 16.2.5 Detection of acetylcholine using the Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217). Each reaction contained 200 µM Amplex® Red re-agent, 1 U/mL HRP, 0.1 U/mL choline oxidase, 0.5 U/mL ace-tylcholinesterase and the indicated amount of acetylcholine in 1X reaction bu�er. Reactions were incubated at room temperature. After 15 and 60 minutes, �uorescence was measured with a �uorescence microplate reader using exci-tation at 560 ± 10 nm and �uorescence detection at 590 ± 10 nm. The inset shows the sensitivity of the 15 min (h) and 60 min (d) assays at low levels of acetylcholine (0–3 µM).

3000

2500

2000

1000

500

0

Acetylcholine (µM)

Fluo

resc

ence

1500

1208040 10060200

60 min

15 min800

400

0��10 2

1200

60 min

15 min

Figure 16.2.4 Detection of electric eel acetylcholines-terase activity using the Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217). Each reaction contained 50 µM acetylcholine, 200 µM Amplex® Red re-agent, 1 U/mL HRP, 0.1 U/mL choline oxidase and the indi-cated amount of acetylcholinesterase in 1X reaction bu�er. Reactions were incubated at room temperature. After 15 and 60 minutes, �uorescence was measured in a �uores-cence microplate reader using excitation at 560 ± 10 nm and �uorescence detection at 590 ± 10 nm. The inset shows the sensitivity of the 15 min (h) and 60 min (d) assays at low levels of acetylcholinesterase activity (0–13 mU/mL).

Figure 16.2.3 Absorption and �uorescence emission spec-tra of resoru�n in pH 9.0 bu�er.







Naloxone Fluorescein for µ-Opioid Receptors�e µ-opioid receptor plays a critical role in analgesia. Among the antagonists that have

been used to de�ne and characterize these receptors are naloxone, a drug used to counteract the e�ects of opioid overdose, and naltrexone, a drug used in the treatment of opioid addic-tion. Naloxone �uorescein (N1384, Figure 16.2.8) has been reported to bind to the µ-opioid binding site with high a�nity,45–47 permitting receptor visualization in transfected Chinese hamster ovary (CHO) cells.48 Flow cytometry analysis of the binding of naloxone �uores-cein to NMDA and µ-opioid receptors (which was displaced by NMDA and met-enkephalin, respectively) has been used to deduce the e�ects of operant conditioning on visual cortex receptor pattern.49

Probes for Amino Acid Neurotransmitter ReceptorsCaged Amino Acid Neurotransmitters

When illuminated with UV light or by multiphoton excitation, caged amino acid neurotrans-mitters are converted into biologically active amino acids that rapidly initiate neurotransmitter action.50,51 �us, these caged probes provide a means of controlling the release—both spatially and temporally—of agonists for kinetic studies of receptor binding or channel opening.

�e di�erent caging groups confer special properties on these photoactivatable probes (Table 5.2). We synthesize two caged versions of L-glutamic acid 52–60 (C7122, G7055), as well as caged carbachol 61,62 (N-(CNB-caged) carbachol, C13654) and caged γ-aminobutyric acid 56,63–66 (O-(CNB-caged) GABA, A7110), all of which are biologically inactive before photolysis.67O-(CNB-caged) GABA (A7110) and γ-(CNB-caged) L-glutamic acid (G7055), which exhibit fast un-caging rates and high photolysis quantum yields, have been used to investigate the activation ki-netics of GABA receptors 66 and glutamate receptors,55 respectively. N-(CNB-caged) L-glutamic acid (C7122) does not hydrolyze in aqueous solution because it is caged on the amino group, thus enabling researchers to use very high concentrations without risk of light-independent glutamic acid production.55,57

Anti–NMDA Receptor AntibodiesN-methyl-D-aspartate (NMDA) receptors constitute cation channels of the central nervous

system that are gated by the excitatory neurotransmitter L-glutamate.68,69 We o�er a�nity-pu-ri�ed rabbit polyclonal antibodies to NMDA receptor subunits 2A, 2B and 2C (A6473, A6474, A6475). �e anti–NMDA receptor subunit 2A and 2B antibodies were generated against fusion proteins containing amino acid residues 1253–1391 of subunit 2A and 984–1104 of subunit 2B, respectively. �ese two antibodies are active against mouse, rat and human forms of the antigens and are speci�c for the subunit against which they were generated. In contrast, the anti–NMDA receptor subunit 2C antibody was generated against amino acid residues 25–130 of subunit 2C and recognizes the 140,000-dalton subunit 2C, as well as the 180,000-dalton subunit 2A and subunit 2B from mouse, rat and human. �ese three a�nity-puri�ed antibodies are suitable for immunohistochemistry 70 (Figure 16.2.9), western blots, enzyme-linked immunosorbent assays (ELISAs) and immunoprecipitations.

Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit�e Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit (A12221) provides an ul-

trasensitive method for continuously detecting glutamic acid 71 or for monitoring glutamate oxidase activity in a �uorescence microplate reader or a �uorometer.72 In this assay, L-glutamic acid is oxidized by glutamate oxidase to produce α-ketoglutarate, NH3 and H2O2. L-Alanine and L-glutamate–pyruvate transaminase are also included in the reaction. �us, the L-glutamic acid is regenerated by transamination of α-ketoglutarate, resulting in multiple cycles of the initial reaction and a signi�cant ampli�cation of the H2O2 produced. Hydrogen peroxide reacts with the Amplex® Red reagent in a 1:1 stoichiometry in a reaction catalyzed by horseradish peroxidase (HRP) to generate the highly �uorescent product resoru�n 73,74 (R363, Section 10.1).

Figure 16.2.7 Muscimol, BODIPY® TMR-X conjugate (M23400).

Figure 16.2.6 BODIPY® FL prazosin (B7433).

Figure 16.2.8 Naloxone �uorescein (N1384).

Figure 16.2.9 Rat brain cryosections labeled with anti-NMDA receptor, subunit 2A (rat), rabbit IgG fraction (A6473) and detected using Alexa Fluor® 488 goat anti–rabbit IgG antibody (A11008). The tissue was also labeled with Alexa Fluor® 594 anti–glial �brillary acidic protein antibody (A21295) and counterstained with TOTO®-3 iodide (T3604), which was pseudocolored light blue in this image.








Figure 16.2.10 Detection of L-glutamic acid using the Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit (A12221). Each reaction contained 50 µM Amplex® Red reagent, 0.125 U/mL HRP, 0.04 U/mL L-glutamate oxidase, 0.25 U/mL L-glutamate–pyruvate transaminase, 100 µM L-alanine and the indicated amount of L-glutamic acid in 1X reaction bu�er. Reactions were incubated at 37°C. After 30 minutes, �uorescence was measured in a �uorescence microplate reader using excitation at 530 ± 12.5 nm and �uorescence detection at 590 ± 17.5 nm.

3,500

3,000

2,500

2,000

1,500

1,000

500

00 5 10 15

Glutamic acid (µM)

Fluo

resc

ence

20

200

150

100

50

00 0.025 0.05 0.075 0.1

Figure 16.2.11 Detection of L-glutamate oxidase using the Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit (A12221). Each reaction contained 50 µM Amplex® Red reagent, 0.125 U/mL HRP, 0.25 U/mL L-glutamate–pyruvate transaminase, 20 µM L-glutamic acid, 100 µM L-alanine and the indicated amount of Streptomyces L-glutamate oxidase in 1X reaction bu�er. Reactions were incubated at 37°C. After 60 minutes, �uorescence was measured in a �uorescence microplate reader using excitation at 530 ± 12.5 nm and �uorescence detection at 590 ± 17.5 nm. The inset represents data from a separate experiment for lower L-glutamate oxidase concentrations and incubation time of 60 minutes (0–1.25 mU/mL).

6,000

5,000

4,000

2,000

1,000

0

Glutamate oxidase (mU/mL)

Fluo

resc

ence

125750 25 50 100

3,000

300

1.21.00.6 0.8

600

1.4

200

400

500

100

00.40.20

REFERENCES1. BMC Neurosci (2009) 10:80; 2. Nano Lett (2008) 8:780; 3. Proc Natl Acad Sci U S A (2007) 104:13666; 4. Am J Physiol Cell Physiol (2006) 290:C728; 5. J Cell Biol (2005) 170:619; 6. J Immunol Methods (2004) 289:169; 7. J Histochem Cytochem (2006) 54:817; 8. Biochemistry (1990) 29:11009; 9. Meth Neurosci (1992) 8:67; 10. J Cell Biol (1998) 141:1613; 11. Proc Natl Acad Sci U S A (1976) 73:4594; 12. J Physiol (1974) 237:385; 13. J Cell Biol (1993) 120:197; 14. J Cell Biol (1993) 120:185; 15. J Neurosci (1995) 15:520; 16. J Cell Biol (1994) 124:139; 17. Biophys J (2006) 90:2192; 18. Proc Natl Acad Sci U S A (2004) 101:17114; 19. J Biol Chem (2008) 283:15160; 20. J Orthop Res (2009) 27:114; 21. J Neurosci (2006) 26:6873; 22. J Clin Invest (2004) 113:265; 23. J Cell Biol (1995) 131:441; 24. J Biol Chem (1993) 268:25108; 25. Proc Natl Acad Sci U S A (1980) 77:4823; 26. Clin Chim Acta (2007) 379:119; 27. J Neurosci (2008) 28:11468; 28. Mol Brain (2008) 1:18; 29. Biochemistry (1979) 18:1875; 30. Toxicon (1991) 29:503; 31. Neuron (1992) 8:353; 32. Science (1998) 281:794; 33. Proc SPIE-Int Soc Opt Eng (2000) 3926:166; 34. Brain Res Dev Brain Res (1997) 102:35; 35. Br J Pharmacol (2009) 158:209; 36. J Pharmacol Exp �er (2009) 331:1118; 37. Br J Pharmacol (2004) 143:899; 38. J Neurosci Res (1992) 31:166; 39. Neural Plast (2000) 7:19; 40. J Neurosci Methods (2008) 171:30; 41. Proc Natl Acad Sci U S A (2007) 104:335; 42. Am J Physiol Renal Physiol (2006) 291:F375; 43. J Neurosci Methods (2005) 143:3; 44. Am J Physiol Renal Physiol (2005) 288:F420; 45. Pharm Res (1986) 3:56; 46. Pharm Res (1985) 6:266; 47. Life Sci (1983) 33 Suppl 1:423; 48. J Neurosci Methods (2000) 97:123; 49. Biol Chem Hoppe Seyler (1995) 376:483; 50. Nat Methods

Because resoru�n has absorption/emission maxima of ~571/585 nm (Figure 16.2.3), there is little interference from auto�uorescence in most biological samples.

If the concentration of L-glutamic acid is limiting in this assay, then the �uorescence increase is proportional to the initial L-glutamic acid concentration. �e Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit allows detection of as little as 10 nM L-glutamic acid in puri�ed systems using a 30-minute reaction time (Figure 16.2.10). If the reaction is modi�ed to include an excess of L-glutamic acid, then this kit can be used to continuously monitor glutamate oxidase activity. For example, puri�ed L-glutamate oxidase from Streptomyces can be detected at levels as low as 40 µU/mL (Figure 16.2.11). �e Amplex® Red reagent has been used to quantitate the activity of glutamate-producing enzymes in a high-throughput assay for drug discovery.71 �e Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit contains:

• Amplex® Red reagent• Dimethylsulfoxide (DMSO)• Horseradish peroxidase (HRP)• H2O2• Concentrated reaction bu�er• L-Glutamate oxidase from Streptomyces sp.• L-Glutamate–pyruvate transaminase from pig heart• L-Glutamic acid• L-Alanine• Detailed protocols

Each kit provides su�cient reagents for approximately 200 assays using a �uorescence mi-croplate reader and a reaction volume of 100 µL per assay.

Probes for Other Receptors�e Molecular Probes® Handbook discusses a diverse array of receptor probes, including

�uorescent derivatives of:

• Low-density lipoprotein (LDL)• Lipopolysaccharides• Epidermal growth factor (EGF)• Transferrin• Fibrinogen• Gelatin and collagen

• Ovalbumin and bovine serum albumin• Casein• Histone H1• Subunit B of cholera toxin• Chemotactic peptide• Insulin

�ese ligands are all transported into the cell by receptor-mediated endocytosis. Additional information about these probes, as well as membrane and �uid-phase markers, can be found in Section 16.1.







DATA TABLE 16.2 PROBES FOR NEUROTRANSMITTER RECEPTORSCat. No. MW Storage Soluble Abs EC Em Solvent NotesA7110 396.28 F,D,LL H2O 262 4500 none pH 7 1, 2A13438 1404.50 F,D,L H2O, DMSO 494 78,000 522 pH 9 3A13439 1586.64 F,D,L H2O, DMSO 491 78,000 516 pH 7 3B1196 ~8400 F,D H2O <300 none 4B1601 7984.14 F H2O <300 see Notes 5B7433 563.41 F,D,L DMSO, EtOH 504 77,000 511 MeOHB13422 9000 F,D,L H2O 495 78,000 519 pH 8 4, 6B13423 9000 F,D,L H2O 593 92,000 617 pH 7 4, 6B35450 9000 F,D,L H2O 649 246,000 668 pH 7 4, 6B35451 9000 F,D,L H2O 554 150,000 567 pH 7 4, 6B35452 9000 F,D,L H2O 680 180,000 704 pH 7 4, 6C7122 326.26 F,D,LL H2O 266 4800 none pH 7 1, 2C13654 439.34 F,D,LL H2O 264 4200 none H2O 1, 2F1176 9000 F,D,L H2O 494 84,000 518 pH 8 4, 6G7055 440.29 F,D,LL H2O, DMSO 262 5100 none pH 7 1, 2M23400 607.46 F,D,L DMSO 543 60,000 572 MeOHN1384 790.84 D,L EtOH, DMF 492 79,000 516 pH 9T1175 9000 F,D,L H2O 553 85,000 577 H2O 4, 6For de�nitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.Notes

1. All photoactivatable probes are sensitive to light. They should be protected from illumination except when photolysis is intended.2. This compound has weaker visible absorption at >300 nm but no discernible absorption peaks in this region.3. The value of EC listed for this peptide conjugate is that of the labeling dye in free solution. Use of this value for the conjugate assumes a 1:1 dye:peptide labeling ratio and no change of EC due to

dye–peptide interactions.4. α-Bungarotoxin, EGF and phallotoxin conjugates have approximately 1 label per peptide.5. This peptide exhibits intrinsic tryptophan �uorescence (Em ~350 nm) when excited at <300 nm.6. The value of EC listed for this α-bungarotoxin conjugate is for the labeling dye in free solution. Use of this value for the conjugate assumes a 1:1 dye:peptide labeling ratio and no change of EC

due to dye–peptide interactions.

PRODUCT LIST 16.2 PROBES FOR NEUROTRANSMITTER RECEPTORSCat. No. Product QuantityA7110 γ-aminobutyric acid, α-carboxy-2-nitrobenzyl ester, tri�uoroacetic acid salt (O-(CNB-caged) GABA) 5 mgA12217 Amplex® Red Acetylcholine/Acetylcholinesterase Assay Kit *500 assays* 1 kitA12221 Amplex® Red Glutamic Acid/Glutamate Oxidase Assay Kit *200 assays* 1 kitA13439 angiotensin II, Alexa Fluor® 488 conjugate 25 µgA13438 angiotensin II, �uorescein conjugate 25 µgA6473 anti-NMDA receptor, subunit 2A (rat), rabbit IgG fraction *a�nity puri�ed* 10 µgA6474 anti-NMDA receptor, subunit 2B (rat), rabbit IgG fraction *a�nity puri�ed* 10 µgA6475 anti-NMDA receptor, subunit 2C (rat), rabbit IgG fraction *a�nity puri�ed* 10 µgB7433 BODIPY® FL prazosin 100 µgB1601 α-bungarotoxin *from Bungarus multicinctus* 1 mgB13422 α-bungarotoxin, Alexa Fluor® 488 conjugate 500 µgB35451 α-bungarotoxin, Alexa Fluor® 555 conjugate 500 µgB13423 α-bungarotoxin, Alexa Fluor® 594 conjugate 500 µgB35450 α-bungarotoxin, Alexa Fluor® 647 conjugate 500 µgB35452 α-bungarotoxin, Alexa Fluor® 680 conjugate 500 µgB1196 α-bungarotoxin, biotin-XX conjugate 500 µgC13654 N-(CNB-caged) carbachol (N-(α-carboxy-2-nitrobenzyl)carbamylcholine, tri�uoroacetic acid salt) 5 mgC7122 N-(CNB-caged) L-glutamic acid (N-(α-carboxy-2-nitrobenzyl)-L-glutamic acid) 5 mgF1176 �uorescein α-bungarotoxin (α-bungarotoxin, �uorescein conjugate) 500 µgG7055 L-glutamic acid, γ-(α-carboxy-2-nitrobenzyl) ester, tri�uoroacetic acid salt (γ-(CNB-caged) L-glutamic acid) 5 mgM23400 muscimol, BODIPY® TMR-X conjugate 1 mgN1384 naloxone �uorescein 5 mgT1175 tetramethylrhodamine α-bungarotoxin (α-bungarotoxin, tetramethylrhodamine conjugate) 500 µg

(2007) 4:619; 51. J Neurosci Methods (2004) 133:153; 52. Nat Neurosci (1998) 1:119; 53. Neuroscience (1998) 86:265; 54. Science (1998) 279:1203; 55. Proc Natl Acad Sci U S A (1994) 91:8752; 56. J Org Chem (1996) 61:1228; 57. Abstr Soc Neurosci (1995) 21:579, abstract 238.11; 58. J Neurosci Methods (1994) 54:205; 59. Science (1994) 265:255; 60. Proc Natl Acad Sci U S A (1993) 90:7661; 61. J Neurosci (2003) 23:9024; 62. Proc

Natl Acad Sci U S A (2000) 97:13895; 63. Methods Enzymol (1998) 291:443; 64. Neuron (1995) 15:755; 65. J Org Chem (1990) 55:1585; 66. J Am Chem Soc (1994) 116:8366; 67. Methods Enzymol (1998) 291:30; 68. Neuron (1994) 12:529; 69. Nature (1991) 354:31; 70. J Neurochem (2000) 75:2040; 71. Anal Biochem (2000) 284:382; 72. Anal Chim Acta (1999) 402:47; 73. Anal Biochem (1997) 253:162; 74. J Immunol Methods (1997) 202:133.

REFERENCES—continued







Section 16.3 Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins

16.3 Probes for Ion Channels and Carriers

Figure 16.3.1 DM-BODIPY® (–)-dihydropyridine (D7443).

Figure 16.3.2 BODIPY® FL verapamil, hydrochloride (B7431).

�is section describes a variety of probes for Ca2+, Na+, K+ and Cl– ion channels and carriers. Chapter 19 and Chapter 21 contain our extensive selection of indicators for these physiologi-cally important ions, providing a means of correlating ion channel activation with subsequent changes in intracellular ion concentration. Ion �ux also a�ects the cell’s membrane potential, which can be measured with the probes described in Chapter 22.

Probes for Ca2+ Channels and CarriersIn both excitable and nonexcitable cells, intracellular Ca2+ levels modulate a multitude of

vital cellular processes—including gene expression, cell viability, cell proliferation, cell motility and cell shape and volume regulation—and thereby play a key role in regulating cell responses to external activating agents. �ese dynamic changes in intracellular Ca2+ levels are regulated by ligand-gated and G-protein–coupled ion channels in the plasma membrane, as well as by mo-bilization of Ca2+ from intracellular stores. One of the best-studied examples of Ca2+-dependent signal transduction is the depolarization of excitable cells, such as those of neuronal, cardiac, skeletal and smooth muscle tissue, which is mediated by inward Ca2+ and Na+ currents. �e Ca2+ current is attributed to the movement of ions through N-, L-, P- and T-type Ca2+ chan-nels, which are de�ned both pharmacologically and by their biophysical properties, including conductance and voltage sensitivity. Here we describe several �uorescent ligands for imaging the spatial distribution and localization of Ca2+ channels in cells, as well as Premo™ Cameleon Calcium Sensor, a genetically encoded, protein-based ratiometric sensor for calcium measure-ments. Our complete selection of Ca2+ indicators is described in Chapter 19.

Fluorescent Dihydropyridine for L-Type Ca2+ Channels�e L-type Ca2+ channel is readily blocked by the binding of dihydropyridine to the chan-

nel’s pore-forming α1-subunit. To facilitate the study of channel number and distribution in single cells, we o�er �uorescent dihydropyridine derivatives. �e high-a�nity (–)-enantiomer of dihydropyridine is available labeled with either the green-�uorescent DM-BODIPY® (D7443, Figure 16.3.1) or the orange-�uorescent ST-BODIPY® (S7445) �uorophore. Knaus and colleagues have shown that these BODIPY® dihydropyridines bind to L-type Ca2+ channels with high af-�nity and inhibit the Ca2+ in�ux in GH3 cells.1–3 For neuronal L-type Ca2+ channels, the (–)-en-antiomers of the DM-BODIPY® dihydropyridine and ST-BODIPY® derivatives each exhibit a Ki of 0.9 nM. �eir a�nities for skeletal muscle L-type Ca2+ channels are somewhat lower. Although DM-BODIPY® dihydropyridine exhibits a more intense �uorescence, the particularly high degree of stereoselectivity retained by the ST-BODIPY® derivatives has proven useful for the in vivo visualization of L-type Ca2+ channels.4 DM-BODIPY® dihydropyridine has proven e�ective as a substrate for functional analysis of ABC drug transporters.5

BODIPY® FL VerapamilLike dihydropyridine, phenylalkylamines also bind to the α1-subunit of L-type Ca2+ chan-

nels and block Ca2+ transport. We o�er a green-�uorescent BODIPY® FL derivative (B7431, Figure 16.3.2) of verapamil, a phenylalkylamine known to inhibit P-glycoprotein–mediated drug e�ux.

�e 170,000-dalton P-glycoprotein is typically overexpressed in tumor cells that have ac-quired resistance to a variety of anticancer drugs (Section 15.6). P-glycoprotein is thought to mediate the ATP-dependent e�ux or sequestration of structurally unrelated molecules, includ-ing actinomycin D, anthracyclines, colchicine, epipodophyllotoxins and vinblastine. Verapamil appears to inhibit drug e�ux by acting as a substrate of P-glycoprotein, thereby overwhelming the transporter’s capacity to expel the drugs. BODIPY® FL verapamil also appears to serve as a substrate for P-glycoprotein. �is �uorescent verapamil derivative preferentially accumulates in the lysosomes of normal, drug-sensitive NIH 3T3 cells but is rapidly transported out of multidrug-resistant cells.6–9Figure 16.3.4 Schematic of the Premo™ Cameleon Calcium

Sensor (P36207, P36208) mechanism.

440 nm

480 nm

+ 4 Ca2+

440 nm

FRET 535 nm







Eosin Derivatives: Inhibitors of the Calcium PumpEosin isothiocyanate (E18) is a potent reversible inhibitor of the erythrocyte plasma mem-

brane calcium pump, with a half-maximal inhibitory concentration of <0.2 µM.10 Eosin isothio-cyanate also reacts irreversibly at the ATP-binding site of this calcium pump. �e succinimi-dyl ester of carboxyeosin diacetate (C22803), a cell membrane–permeant eosin derivative, also inhibits the erythrocyte plasma membrane Ca2+ pump.11,12 Fluorescein isothiocyanate (F143, Section 1.4) is a weaker inhibitor of the erythrocyte plasma membrane calcium pump.

Premo™ Cameleon Calcium Sensor�e Premo™ product line combines genetically encoded ion indicators and environmental

sensors with e�cient BacMam delivery (BacMam Gene Delivery and Expression Technology—Note 11.1) for intracellular measurements in mammalian cells. Premo™ Cameleon Calcium Sensor (P36207, P36208) is a ratiometric calcium-sensitive �uorescent protein that is delivered by BacMam baculovirus-mediated transduction to a variety of mammalian cell types. �is content and delivery system provides an e�ective and robust technique for measuring Ca2+ mobilization in transduced cells using microplate assays or �uorescence microscopy (Figure 16.3.3).

�e Premo™ Cameleon Calcium Sensor is based on the YC3.60 version of the �uorescent protein (FP)–based sensor (cameleon) family developed by Tsien, Miyawaki and co-workers, which is reported to have a Ca2+ dissociation constant of 240 nM.13,14 �e sensor comprises two �uorescent proteins (Enhanced Cyan Fluorescent Protein or ECFP and Venus variant of Yellow Fluorescent Protein or YFP), linked by the calmodulin-binding peptide M13 and calmodulin. Upon binding four calcium ions, calmodulin undergoes a conformational change by wrapping itself around the M13 peptide, which changes the e�ciency of the �uorescence resonance energy transfer (FRET) between the CFP donor and the YFP acceptor �uorophores (Figure 16.3.4). Following this conformational change, there is an increase in YFP emission (525–560 nm) and a simultaneous decrease in CFP emission (460–500 nm) (Figure 16.3.5), making Cameleon an e�ective reporter of calcium mobilization. �e ratiometric readout of the Premo™ Cameleon Calcium Sensor—an increase in YFP emission (535 nm, green-yellow emission) and a decrease in CFP emission (485 nm, blue emission)—reduces assay variations due to compound or cellular auto�uorescence, nonuniform cell plating, di�erences in expres-sion levels between cells, instability of instrument illumination and changes in illumination pathlength.

�e Premo™ Cameleon Calcium Sensor is designed to readily and accurately detect intracel-lular calcium �ux from di�erent receptors. Standard pharmacological assays for multiple GPCR agonists and antagonists have been tested. An example of the robustness and reproducibility and accuracy of the system is demonstrated using the endogenous histamine receptor in conjunction with histamine, pyrilamine, and thioperamide in HeLa cells (Figure 16.3.6). Expression levels will be maintained for several days, enabling iterative assays to be run, for instance, when exam-ining agonist/antagonist relationships on the same cells.

Figure 16.3.5 Fluorescence emission spectra of Premo™ Cameleon Calcium Sensor (P36207, P36208). The dashed line indicates the spectra in the absence of Ca2+; the solid line shows the �uorescence resonance energy transfer (FRET)–based change upon Ca2+ binding.

Wavelength (nm)

450 500 550 600

Fluo

resc

ence

em

issi

on

Ex = 435 nm

Figure 16.3.6 Agonist and antagonist dose response curves. HeLa cells were plated in a 96-well plate at a den-sity of 15,000 cells/well, transduced with Premo™ Cameleon Calcium Sensor (P36207, P36208), and incubated overnight at 37°C. The following day, a histamine dose response was performed (A). A separate plate was used to evaluate an antagonist dose response with pyrilamine (j) and thiop-eramide (m) in the presence of an EC80 concentration of histamine (B). Pyrilamine is a known H1 receptor antagonist that couples through Gq proteins and the second messen-ger Ca2+. Thioperamide is a known H3 receptor antagonist that couples through Gi proteins and the second messen-ger cAMP.

Histamine (µM)

0.01 1010.1 100

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/CFP

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io (m

ax–m

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Figure 16.3.3 Porcine left atrial appendage progenitor cells were transfected with Premo™ Cameleon calcium sensor (P36207, P36208); ATP (20 µM �nal concentration) was applied to the cells the following day and the cells were imaged using a Zeiss 5 Live high-speed confocal system (Carl Zeiss MicroImaging). Excitation was with a 405 nm diode laser (50 mw) operated at 50% power. Emission was collected simultaneously on two linear CCD detectors using a 490 nm dichroic mirror to split the beam through a 415–480 nm bandpass �lter for CFP and a 550 nm longpass �lter for YFP. Images were collected at a rate of 10 frames per second (512 x 512 pixels) using a 40x Plan-Neo�uar 1.3 NA oil immersion objective lens.








Probes for Na+ Channels and CarriersAmiloride Analogs: Probes for the Na+ Channel and the Na+/H+ Antiporter

Amiloride is a compound known to inhibit the Na+/H+ antiporter of vertebrate cells by act-ing competitively at the Na+-binding site.15 �e antiporter extrudes protons from cells using the inward Na+ gradient as a driving force, resulting in intracellular alkalinization. In 1967, Cragoe and co-workers reported the synthesis of amiloride and several amiloride analogs, which are pyr-azine diuretics that inhibit the Na+ channel in urinary epithelia.16 Since then, more than 1000 dif-ferent amiloride analogs have been synthesized and many of these tested for their speci�city and potency in inhibiting the Na+ channel, Na+/H+ antiporter and Na+/Ca2+ exchanger.17 Unmodi�ed amiloride inhibits the Na+ channel with an IC50 of less than 1 µM. Additionally, amiloride is an important tool for studying the Na+/H+ antiporter. Structure–activity relationships have dem-onstrated that amiloride analogs with hydrophobic groups in the drug are the most potent and speci�c inhibitors for the Na+/H+ antiporter.17–22 For example, 5-(N-ethyl-N-isopropyl)amiloride (EIPA, E3111; Figure 16.3.7) is 200-fold more potent than amiloride for inhibiting this antiporter.

Ouabain Probes for Na+/K+-ATPaseOuabain is a member of a class of glycosylated steroids collectively known as cardiac gly-

cosides due to their therapeutic e�cacy in the treatment of congestive heart failure. Ouabain achieves this e�ect by binding to the catalytic α-subunit of Na+/K+-ATPase and inhibiting its transport of Na+ across the plasma membrane. 9-Anthroyl ouabain (A1322) is useful for lo-calizing Na+/K+-ATPase and for studying its membrane orientation, mobility and dynamics.23 Anthroyl ouabain has also been employed to investigate Na+/K+-ATPase’s active site, inhibition and conformational changes,24–29 as well as to investigate the kinetics of cardiac glycoside bind-ing.30–35 BODIPY® FL ouabain (B23461, Figure 16.3.8) has been used in combination with Alexa Fluor® 555 cholera toxin B (C22843, Section 16.1) for visualizing Na+/K+-ATPase and ganglioside GM1 domain localization in lymphocyte plasma membranes.36

Using BacMam Technology to Deliver and Express Sodium Channel cDNASodium channel cDNAs that have been engineered into a baculovirus gene delivery/expres-

sion system using BacMam technology (BacMam Gene Delivery and Expression Technology—Note 11.1) are also available, including the Nav1.2 cDNA (B10341) and the Nav1.5 cDNA (B10335).

�e BacMam system uses a modi�ed insect cell baculovirus as a vehicle to e�ciently deliver and express genes in mammalian cells with minimum e�ort and toxicity. �e use of BacMam delivery in mammalian cells is relatively new, but well described, and has been used extensively in a drug discovery setting.37 Furthermore, constitutively expressed ion channels and other cell surface proteins have been shown to contribute to cell toxicity in some systems, and may be subject to clonal dri� and other inconsistencies that hamper successful experimentation and screening. �us, transient expression systems such as the BacMam gene delivery and expression system are increasingly methods of choice to decrease variability of expression in such assays.

U2OS cells (ATCC number HTB-96) have been shown to demonstrate highly e�cient ex-pression of BacMam delivered targets in a null background ideal for screening in a heterologous expression system. �e U2OS cell line is recommended for use if your particular cell line does not e�ciently express the BacMam targets. Examples of other cell lines that are e�ciently transduced by BacMam technology include HEK 293, HepG2, BHK, Cos-7 and Saos-2.

Probes for K+ Channels and CarriersGlibenclamide Probes for the ATP-Dependent K+ Channel

Glibenclamide blocks the ATP-dependent K+ channel, thereby eliciting insulin secretion.38 We have prepared the green-�uorescent BODIPY® FL glibenclamide (BODIPY® FL glyburide, E34251; Figure 16.3.9) and red-�uorescent BODIPY® TR glibenclamide (BODIPY® TR glyburide, E34250) as probes for the ATP-dependent K+ channel. BODIPY® TR glibenclamide has been used to detect sulfonylurea receptors associated with ATP-dependent K+ channels in bovine monocytes and in β-cells 39,40 and to label a novel mitochondrial ATP-sensitive potassium channel in brain.41

�e sulfonylurea receptors of ATP-dependent K+ channels are prominent on the endoplas-mic reticulum (ER). �erefore, because these probes are also e�ective live-cell stains for ER,

Figure 16.3.8 BODIPY® FL ouabain (B23461).

Figure 16.3.7 5-(N-ethyl-N-isopropyl)amiloride, hydrochlo-ride (E3111).

Figure 16.3.9 ER-Tracker™ Green (BODIPY® FL gliben-clamide, E34251).







BODIPY® FL glibenclamide and BODIPY® TR glibenclamide are also referred to as ER-Tracker™ Green and ER-Tracker™ Red, respectively; see Section 12.4 for a description of this application. Variable expression of sulfonylurea receptors in some specialized cell types may result in non-ER labeling with these probes.

FluxOR™ Potassium Ion Channel Assay�e FluxOR™ Potassium Ion Channel Assay Kits (F10016, F10017)

provide a �uorescence-based assay for high-throughput screening of potassium ion channel and transporter activities.42,43 �e FluxOR™ Potassium Ion Channel Assay Kits take advantage of the well-described permeability of potassium channels to thallium (Tl+) ions. When thallium is present in the extracellular solution containing a stimulus to open po-tassium channels, channel activity is detected with a cell-permeant thal-lium indicator dye that reports large increases in �uorescence emission at 525 nm as thallium �ows down its concentration gradient and into the cells (Figure 16.3.10). In this way, the �uorescence reported in the FluxOR™ system becomes a surrogate indicator of activity for any ion channel or transporter that is permeable to thallium, including the human ether-a-go-go–related (hERG) channel, one of the human cardiac potassium channels. �e FluxOR™ potassium ion channel assay has been validated for homogeneous high-throughput pro�ling of hERG channel inhibition using BacMam-mediated transient expression of hERG.42 �e FluxOR™ Potassium Ion Channel Assay Kits can also be used to study potassium

Figure 16.3.10 Thallium redistribution in the FluxOR™ assay. Basal �uorescence from cells loaded with FluxOR™ reagent (provided in the FluxOR™ Potassium Ion Channel Assay Kits; F10016, F10017) is low when potassium channels remain unstimulated, as shown in the left panel. When thallium is added to the assay with the stimulus, the thallium �ows down its concentration gradient into the cells, activating the dye as shown in the right panel.

Ion channel Ion channel

Tl +

Tl+ Tl +

Tl+

Tl+

Tl+

Tl+

Tl+

Tl +

Closed

Open Tl +

Tl+

Tl+

Tl +

Tl+

StimulatedResting

Thallium Dye

Extracellular Intracellular Extracellular Intracellular

Tl +

Tl+

Tl+

Tl+

Figure 16.3.11 FluxOR™ potassium ion channel assays (F10016, F10017) performed on fresh and frozen U2OS cells transduced with the BacMam hERG reagent (B10019, B10033). A) Raw data ob-tained in the FluxOR™ assay determination of thallium �ux in U2OS cells transduced with BacMam-hERG and kept frozen until the day of use. The arrow indicates the addition of the thallium/po-tassium stimulus, and upper and lower traces indicate data taken from the minimum and maximum doses of cisapride used in the determination of the dose-response curves. B) Raw pre-stimulus peak and baseline values were boxcar averaged and normalized, and indicate the fold increase in �uorescence over time. C) Data generated in a dose-response determination of cisapride block on BacMam hERG expressed in U2OS cells freshly prepared from overnight expression after viral transduction. D) Parallel data obtained from cells transduced with BacMam-hERG, stored for 2 weeks in liquid nitrogen, thawed, and plated 4 hours prior to running the assay. Error bars indicate standard deviation, n = 4 per determination.

39

34

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A B C D

co-transport processes that accommodate the transport of thallium into cells.44 Furthermore, resting potassium channels and inward recti�er po-tassium channels like Kir2.1 can be assayed by adding stimulus bu�er with thallium alone, without any depolarization to measure the signal.

�e FluxOR™ reagent, a thallium indicator dye, is loaded into cells as a membrane-permeable AM ester. Loading is assisted by the propri-etary PowerLoad™ concentrate, an optimized formulation of nonionic Pluronic® surfactant polyols that act to disperse and stabilize AM ester dyes for optimal loading in aqueous solution. �is PowerLoad™ con-centrate is also available separately (P10020) to aid the solubilization of water-insoluble dyes and other materials in physiological media.

Once inside the cell, the non�uorescent AM ester of the FluxOR™ dye is cleaved by endogenous esterases into a weakly �uorescent (basal �uorescence), thallium-sensitive indicator. �e thallium-sensitive form is retained in the cytosol, and its extrusion is inhibited by water-soluble probenecid (P36400, Section 19.8), which blocks organic anion pumps. For most applications, cells are loaded with the dye at room tempera-ture. For best results, the dye-loading bu�er is then replaced with fresh, dye-free assay bu�er (composed of physiological HBSS containing pro-benecid), and cells are ready for the high-throughput screening assay.

Each FluxOR™ Potassium Ion Channel Assay Kit contains:

• FluxOR™ reagent• FluxOR™ assay bu�er• PowerLoad™ concentrate• Probenecid• FluxOR™ chloride-free bu�er• Potassium sulfate (K2SO4) concentrate• �allium sulfate (Tl2SO4) concentrate• Dimethylsulfoxide (DMSO)• Detailed protocols

�e FluxOR™ Kits provide a concentrated thallium solution along with su�cient dye and bu�ers to perform ~4000 (F10016) or ~40,000 (F10017) assays in a 384-well microplate format. �ese kits allow maxi-mum target �exibility and ease of operation in a homogeneous format. �e FluxOR™ potassium ion channel assay has been demonstrated for use with CHO and HEK 293 cells stably expressing hERG, as well as U2OS cells transiently transduced with BacMam hERG reagent 42 (B10019, B10033) (Figure 16.3.11). More information is available at www.invitrogen.com/handbook/�uxorpotassium.








Using BacMam Technology to Deliver and Express Potassium Channel cDNA

Potassium channel cDNAs that have been engineered into a bacu-lovirus gene delivery/expression system using BacMam technology (BacMam Gene Delivery and Expression Technology—Note 11.1) are also available for use with the FluxOR™ Potassium Ion Channel Assay Kits, including the human ether-a-go-go related gene (hERG) (Figure 16.3.12), several members of the voltage-gated K+ channel (Kv) gene family and two members of the inwardly rectifying K+ channel (Kir) gene family:

• BacMam hERG 42 (for 10 microplates, B10019; for 100 microplates, B10033)

• BacMam Kv1.1 (for 10 microplates, B10331)• BacMam Kv1.3 (for 10 microplates, B10332)• BacMam Kv2.1 (for 10 microplates, B10333)• BacMam Kv7.2 and Kv7.3 (for 10 microplates, B10147)• BacMam Kir1.1 (for 10 microplates, B10334)• BacMam Kir2.1 (for 10 microplates, B10146)

�e BacMam system uses a modi�ed insect cell baculovirus as a vehicle to e�ciently deliver and express genes in mammalian cells with minimum e�ort and toxicity. �e use of BacMam delivery in mam-malian cells is relatively new, but well described, and has been used extensively in a drug discovery setting.37 Furthermore, constitutively expressed ion channels and other cell surface proteins have been shown to contribute to cell toxicity in some systems, and may be subject to clonal dri� and other inconsistencies that hamper successful experi-mentation and screening. �us, transient expression systems such as BacMam technology are increasingly methods of choice to decrease variability of expression in such assays.

Figure 16.3.12 BacMam-hERG gene delivery and expression. This schematic depicts the mechanism of BacMam-mediated gene delivery into a mammalian cell and expression of the hERG gene (B10019, B10033). The hERG gene resides within the baculoviral DNA, down-stream of a CMV promoter that drives its expression when introduced into a mammalian target cell. BacMam viral particles are taken up by endocytic pathways into the cell, and the DNA within them is released for transcription and expression. The translated protein is then folded for insertion into the membrane, forming functional hERG ion channels. This process begins within 4–6 hours and in many cell types is completed after an overnight period.

Promoter human Ether-à-go-go Related Gene

Baculovirus

hERG Gene

Endocytotic entry

DNA movesto nucleus hERG gene

transcribed

DNA

mRNA

mRNAtranslated

Assembly

Ion channel

Membraneinsertion

Assembly

Figure 16.3.13 DIDS (4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid, disodium salt, D337).

CH CH � C S�CS

S�� S

��

Figure 16.3.14 Bis-(1,3-dibutylbarbituric acid)pentamethine oxonol (DiBAC4(5), B436).

U2OS cells (ATCC number HTB-96) have been shown to demon-strate highly e�cient expression of BacMam-delivered targets in a null background ideal for screening in a heterologous expression system. �e U2OS cell line is recommended for use if your particular cell line does not e�ciently express the BacMam targets. Examples of other cell lines that are e�ciently transduced by BacMam technology include HEK 293, HepG2, BHK, Cos-7 and Saos-2.

Probes for Anion TransportersStilbene Disulfonates: Anion-Transport Inhibitors

We o�er three stilbene disulfonates that have been employed to inhibit (frequently irreversibly) anion transport 45 in a large number of mammalian cell types:

• DIDS (D337, Figure 16.3.13)• H2DIDS (D338)• DNDS (D673)

Our stilbene disulfonate probes, which are 95–99% pure by HPLC, have signi�cantly higher purity and more de�ned composition than those available from other commercial sources. DNDS was among the inhibitors used to characterize three di�erent anion exchangers in the membranes of renal brush border cells and to compare these exchangers with the band-3 anion-transport protein of erythrocyte membranes.46

�ese stilbene disulfonates can, in some cases, bind speci�cally to proteins that are not anion transporters. For example, DIDS and H2DIDS complex speci�cally with the CD4 glycoprotein on T-helper lymphocytes and macrophages, blocking HIV type-1 growth at mul-tiple stages of the virus life cycle.47







DiBAC4(5)�e membrane potential–sensing dye bis-(1,3-dibutylbarbituric

acid)pentamethine oxonol (DiBAC4(5), B436; Figure 16.3.14) initially inhibits Cl– exchange with an IC50 of 0.146 µM. However, this inhibition increases with time to an IC50 of 1.05 nM, making DiBAC4(5) a more potent inhibitor than DIDS, which has an IC50 of 31 nM under similar conditions.48

Eosin MaleimideAlthough usually selectively reactive with thiols, eosin-5-ma-

leimide (E118, Section 2.2) is known to react with a speci�c lysine residue of the band-3 protein in human erythrocytes, inhibiting anion exchange in these cells and providing a convenient tag for observing band-3 behavior in the membrane.49–51 Eosin-5-isothiocyanate (E18) has similar reactivity with band-3 proteins.52,53

Premo™ Halide Sensor�e �uorescent protein–based Premo™ Halide Sensor (P10229)

is a pharmacologically relevant sensor for functional studies of li-gand- and voltage-gated chloride channels and their modulators in cells. Chloride channels are involved in cellular processes as critical and diverse as transepithelial ion transport, electrical excitability, cell volume regulation and ion homeostasis. Given their physiological sig-ni�cance, it follows that defects in their activity can have severe im-plications, including such conditions as cystic �brosis and neuronal degeneration. �us, chloride channels represent important targets for drug discovery.54 Other methods for detecting chloride are described in Section 21.2.

Premo™ Halide Sensor combines a Yellow Fluorescent Protein (YFP) variant sensitive to halide ions with the e�cient and noncyto-pathic BacMam delivery and expression technology (BacMam Gene Delivery and Expression Technology—Note 11.1). Premo™ Halide Sensor is based on the Venus variant of Aequorea victoria Green

Figure 16.3.15 Principle of Premo™ Halide Sensor Sensor (P10229): Iodide redistribution upon chloride channel activation. Basal �uorescence from Premo™ Halide Sensor is high when chloride channels are closed or blocked. Upon activation (opening) of chloride chan-nels, the iodide ions enter the cell, down its concentration gradient, and quench the �uores-cence from Premo™ Halide Sensor.

Ion channel Ion channelClosed

Open

ActivatedResting

IodidePremo™

Halide Sensor

Extracellular Intracellular Extracellular Intracellular

Figure 16.3.16 Quenching of Premo™ Halide Sensor �uorescence by increasing concen-trations of iodide and chloride. U2OS cells were transduced with Premo™ Halide Sensor. After 24 hours, cells were trypsinized and lysed by resuspension in sterile distilled water. Fluorescence quenching of the lysate was examined using increasing concentrations of NaCl (A) and NaI (B). Iodide induces substantially greater quenching of Premo™ Halide Sensor �uo-rescence than chloride.

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A B

Fluorescent Protein (GFP), which displays enhanced �uorescence, in-creased folding, and reduced maturation time when compared with YFP.55 Additional mutations H148Q and I152L were made within the Venus sequence to increase the sensitivity of the Venus �uorescent protein to changes in local halide concentration, in particular iodide ions.56 Because chloride channels are also permeable to the iodide ion (I), iodide can be used as a surrogate for chloride. Upon stimulation, a chloride channel or transporter opens and iodide �ows down the con-centration gradient into the cells, where it quenches the �uorescence of the expressed Premo™ Halide Sensor protein (Figure 16.3.15). �e decrease in Premo™ Halide Sensor �uorescence is directly proportional to the ion �ux, and therefore the chloride channel or transporter ac-tivity. Premo™ Halide Sensor shows an excitation and emission pro�le similar to YFP (Figure 16.3.16) and can be detected using standard GFP/FITC or YFP �lter sets. Halide-sensitive YFP-based constructs in conjunction with iodide quenching have been used in high-throughput screening (HTS) to identify modulators of calcium-activated chloride channels.57

Premo™ Halide Sensor (P10229) is prepackaged and ready for im-mediate use. It contains all components required for cellular delivery and expression, including baculovirus carrying the genetically encod-ed biosensor, BacMam enhancer and stimulus bu�er. Premo™ Halide Sensor has been demonstrated to transduce multiple cell lines including BHK, U2OS, HeLa, CHO, and primary human bronchial epithelial cells (HBEC), providing the �exibility to assay chloride-permeable channels in a wide range of cellular models. To uncouple cell maintenance and preparation from cell screening, BacMam-transduced cells can be di-vided into aliquots and frozen for later assay. Both stable cell lines and human primary cells can be prepared frozen and “assay-ready” and can be subsequently plated as little as 4 hours prior to screening. Screening can be conducted in complete medium and without any wash steps. Chloride channel assays with Premo™ Halide Sensor are compatible with standard �uorescence HTS platforms.








REFERENCES

PRODUCT LIST 16.3 PROBES FOR ION CHANNELS AND CARRIERSCat. No. Product Quantity

A1322 9-anthroyl ouabain 5 mgB10334 BacMam Kir1.1 *for 10 microplates* 1 kitB10146 BacMam Kir2.1 *for 10 microplates* 1 kitB10331 BacMam Kv1.1 *for 10 microplates* 1 kitB10332 BacMam Kv1.3 *for 10 microplates* 1 kitB10333 BacMam Kv2.1 *for 10 microplates* 1 kitB10147 BacMam Kv7.2 and Kv7.3 *for 10 microplates* 1 kitB10341 BacMam Nav1.2 *for 10 microplates* 1 kitB10335 BacMam Nav1.5 *for 10 microplates* 1 kitB10019 BacMam-hERG *for 10 microplates* 1 kitB10033 BacMam-hERG *for 100 microplates* 1 kitB436 bis-(1,3-dibutylbarbituric acid)pentamethine oxonol (DiBAC4(5)) 25 mgB23461 BODIPY® FL ouabain 100 µgB7431 BODIPY® FL verapamil, hydrochloride 1 mgC22803 CEDA, SE (5-(and-6)-carboxyeosin diacetate, succinimidyl ester) *mixed isomers* 5 mgD337 DIDS (4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid, disodium salt) 100 mgD338 4,4’-diisothiocyanatodihydrostilbene-2,2’-disulfonic acid, disodium salt (H2DIDS) 100 mgD7443 DM-BODIPY® (–)-dihydropyridine *high a�nity enantiomer* 25 µgD673 DNDS (4,4’-dinitrostilbene-2,2’-disulfonic acid, disodium salt) 1 gE18 eosin-5-isothiocyanate 100 mgE34251 ER-Tracker™ Green (BODIPY® FL glibenclamide) *for live-cell imaging* 100 µgE34250 ER-Tracker™ Red (BODIPY® TR glibenclamide) *for live-cell imaging* 100 µgE3111 5-(N-ethyl-N-isopropyl)amiloride, hydrochloride 5 mgF10016 FluxOR™ Potassium Ion Channel Assay *for 10 microplates* 1 kitF10017 FluxOR™ Potassium Ion Channel Assay *for 100 microplates* 1 kitP10020 PowerLoad™ concentrate, 100X 5 mLP36207 Premo™ Cameleon Calcium Sensor *for 10 microplates* 1 kitP36208 Premo™ Cameleon Calcium Sensor *for 100 microplates* 1 kitP10229 Premo™ Halide Sensor *for 10 microplates* 1 kitS7445 ST-BODIPY® (-)-dihydropyridine *high a�nity enantiomer* 25 µg

DATA TABLE 16.3 PROBES FOR ION CHANNELS AND CARRIERSCat. No. MW Storage Soluble Abs EC Em Solvent NotesA1322 788.89 F,D,L DMSO 362 7500 471 MeOHB436 542.67 L DMSO, EtOH 590 160,000 616 MeOH 1B7431 769.18 F,D,L DMSO, EtOH 504 74,000 511 MeOHB23461 858.74 F,D,L DMSO 503 80,000 510 MeOHC22803 873.05 F,D DMSO <300 noneD337 498.47 F,DD H2O 341 61,000 415 H2O 2D338 500.48 F,DD H2O 286 41,000 none MeOH 2D673 474.32 L H2O 352 32,000 none H2OD7443 686.48 F,D,L,A DMSO, EtOH 504 83,000 511 MeOHE18 704.97 F,DD,L pH >6, DMF 521 95,000 544 pH 9 2E3111 336.22 D,L H2O, MeOH 378 23,000 423 MeOHE34250 915.23 F,D,L DMSO, H2O 587 60,000 615 MeOHE34251 783.10 F,D,L DMSO, H2O 504 76,000 511 MeOHS7445 760.57 F,D,L,A DMSO, EtOH 565 143,000 570 MeOHFor de�nitions of the contents of this data table, see “Using The Molecular Probes® Handbook” in the introductory pages.Notes

1. Oxonols may require addition of a base to be soluble.2. Isothiocyanates are unstable in water and should not be stored in aqueous solution.

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