CHAPTER 1 CHAPTER 14 Fluorophores and Fluorescent …...Section 14.1 Choosing a Tracer ˜˚.˜ Choosing a Tracer Figure 14.1.1Collage of images of cyanobacteria stained with various

CHAPTER 14

Fluorescent Tracers of Cell Morphology and Fluid Flow

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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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.

FO

URT

EEN

CHAPTER 14

Fluorescent Tracers of Cell Morphology and Fluid Flow

14.1 Choosing a Tracer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 593

14.2 Membrane-Permeant Reactive Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595Thiol-Reactive CellTracker™ Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595

CellTracker™ Probes in a Variety of Fluorescent Colors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596

Applications for CellTracker™ Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 596

Other Thiol-Reactive Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597

Chloromethyl Derivatives of SNARF®-1 and H2DCFDA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597

Bimanes: Blue-Fluorescent Reactive Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597

Amine-Reactive Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597

CFSE and Its Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 597

CellTrace™ Violet Cell Proliferation Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598

CellTrace™ Oregon Green® 488 Carboxylic Acid Diacetate Succinimidyl Ester. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598

CellTrace™ Far Red DDAO-SE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 598

Amine-Reactive SNARF®-1 Carboxylic Acid Acetate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599

Data Table 14.2 Membrane-Permeant Reactive Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599

Product List 14.2 Membrane-Permeant Reactive Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600

14.3 Polar Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600Fixable Polar Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600

Alexa Fluor® Hydrazides and Hydroxylamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 600

Other Alexa Fluor® Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602

Lucifer Yellow CH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602

Other Lucifer Yellow Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602

Cascade Blue® Hydrazide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602

Other Cascade Blue® and Alexa Fluor® 405 Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604

Biocytin and Other Biotin Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604

Fluorescent Biotin Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604

Non�xable Polar Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604

Fluorescein Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 604

Cell-Permeant Fluorescein Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 606

Fluorescein Substitutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607

Sulforhodamines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607

Hydroxycoumarins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607

Polysulfonated Pyrenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608

ANTS–DPX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608

Lanthanide Chelates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608

TOTO®, YOYO® and SYTO® Nucleic Acid Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 608

Caged Fluorescent Dye Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609

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.

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Chapter 14 — Fluorescent Tracers of Cell Morphology and Fluid Flow

Fluorescent Retrograde Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609

True Blue and Nuclear Yellow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609

Hydroxystilbamidine and Aminostilbamidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610

Propidium Iodide and DAPI for Retrograde Tracing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610

NeuroTrace® Fluorescent Nissl Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610

Polar Spin Label . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611

Signal Ampli�cation of Polar Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612

In�ux™ Pinocytic Cell-Loading Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612

Loading P2X7 Receptor–Expressing Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613

Data Table 14.3 Polar Tracers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615

Product List 14.3 Polar Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616

14.4 Tracers for Membrane Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618Long-Chain Carbocyanines: DiI, DiO and Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618

DiI, DiO, DiD and DiR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618

Vybrant® DiI, DiO and DiD Cell-Labeling Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 619

NeuroTrace® DiI, DiO and DiD Tissue-Labeling Pastes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620

FAST DiI™ and FAST DiO™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620

CM-DiI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 620

Sulfonated Carbocyanines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 621

Other DiO and DiI Analogs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622

Neuronal Tracing Studies with DiI and DiO Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622

Long-Term Cell Tracing with Membrane Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622

Tissue Processing and Electron Microscopy with Long-Chain Carbocyanines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622

DiA and FAST DiA™ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 622

Lipophilic Tracer Sampler Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623

Octadecyl Rhodamine B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623

Plasma Membrane Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623

CellMask™ Plasma Membrane Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623

Fluorescent Protein–Based Plasma Membrane Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623

FM® and RH Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623

Image-iT® LIVE Plasma Membrane and Nuclear Labeling Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624

Vybrant® Lipid Raft Labeling Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625

Image-iT® LIVE Intracellular Membrane and Nuclear Labeling Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626

FluoroMyelin™ Fluorescent Myelin Stains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 626

BrainStain™ Imaging Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 627

Data Table 14.4 Tracers for Membrane Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628

Product List 14.4 Tracers for Membrane Labeling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629

14.5 Fluorescent and Biotinylated Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630Properties of Molecular Probes® Dextran Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630

A Wide Selection of Substituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630

Dextran Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630

Degree of Substitution of Molecular Probes® Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630

Dextran Net Charge and Method of Substitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630

Dextran Fixability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630


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

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Loading Cells with Dextrans and Subsequent Tissue Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631

Neuronal Tracing with Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631

Multilabeled Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632

3000 MW Dextrans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 632

NeuroTrace® BDA-10,000 Neuronal Tracer Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633

Cell Lineage Tracing with Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634

Studying Intercellular Communication with Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634

Probing Membrane Permeability with Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635

Following Endocytosis with Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635

Fluorescent Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635

pH Indicator Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635

Tracing Fluid Transport with Dextrans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636

Product List 14.5 Fluorescent and Biotinylated Dextrans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636

14.6 Microspheres and Qdot® Nanocrystals for Tracing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 638Fluorescent Microspheres for Regional Blood Flow Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 638

FluoSpheres® Microspheres for Blood Flow Determination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 638

FluoSpheres® Color Kits for Regional Blood Flow Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639

Fluorescent Microsphere Resource Center . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639

Particle and Cell Tracking with Fluorescent Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639

FluoSpheres® Beads with 0.04 µm Diameters for Microinjection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639

FluoSpheres® Beads with 1.0 µm Diameters for Tracer Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639

SAIVI™ 715 Injectable Contrast Agents with 0.1 µm and 2 µm Diameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640

Europium and Platinum Luminescent Microspheres for Time-Resolved Fluorometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640

Microspheres for Monitoring Air�ow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641

Transplantation and Migration Studies with Fluorescent Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641

Neuronal Tracing with Fluorescent Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641

Following Phagocytosis with Fluorescent Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641

Tracking the Movement of Proteins and Other Biomolecules with Fluorescent Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641

Qdot® Nanocrystal Tracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 642

Qtracker® Cell Labeling Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 642

Qtracker® Non-Targeted Quantum Dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643

Product List 14.6 Microspheres and Qdot® Nanocrystals for Tracing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 644

14.7 Protein Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645Albumin Conjugates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645

Fluorescent BSA, Ovalbumin and Parvalbumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645

Dinitrophenylated Bovine Serum Albumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645

DQ™ BSA, DQ™ Ovalbumin and DQ™ Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 645

Injectable Contrast Reagents for Small Animal in vivo Imaging Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646

Casein Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646

PHA-L: An Important Anterograde Tracer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646

Cholera Toxin Subunit B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646

Fluorescent Protein–Based Cytosol Marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647

Phycobiliproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647

Alexa Fluor® 488 Soybean Trypsin Inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 647

Product List 14.7 Protein Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 648



thermofisher.com/probes




MitoTracker® Deep Red 633, Alexa Fluor® 568 goat anti–rabbit IgG, Alexa Fluor® 488 phalloidin and Hoechst 33258.



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Section 14.1 Choosing a Tracer

14.1 Choosing a Tracer

Figure 14.1.1 Collage of images of cyanobacteria stained with various blue- or green-�uo-rescent probes to complement the natural red auto�uorescence from chlorophyll and phy-cobilisomes. The round cells are Synechocystis sp. (strain PCC 6803), and their membranes were labeled with green-�uorescent BODIPY® FL propionic acid (D2183). The cylindrical cells are Synechococcus sp. (strain PCC 7942), stained with blue-�uorescent DAPI (D1306, D3571, D21490). The �lamentous cyanobacteria, Anabaena cylindrica, were labeled with either the green-�uorescent cystosolic stain, CellTracker™ Green BODIPY® (C2102), or with the lipophilic membrane stain BODIPY® FL (D2183). The image was contributed by Mary Sarcina, University College, London.

To serve as an e�ective tracer of cell morphology, a �uorescent probe or other detectable molecule must have the capacity for localized introduction into a cell or organelle, as well as for long-term retention within that structure. If used with live cells and tissues, then the tracer should also be biologically inert and nontoxic. When these conditions are satis�ed, the �uorescence or other detectable properties of the tracer can be used to track the position of the tracer over time. Fluorescent tracers can be employed to investigate �ow in capillaries, to de�ne neuronal cell connectivity and to study dye translocation through gap junctions, as well as to follow cell division, cell lysis or liposome fu-sion. Furthermore, they can be used to track the movements of labeled cells in culture, tissues or intact organisms. �e review of techniques for tracing neuronal pathways by Bohland and co-workers 1 is particularly recommended.

Although the predominant tracers have been �uorescent, not all of the useful tracers are intrinsically detectable. For example, biotin deriv-atives are widely used as polar tracers, especially in neurons. However, when a biotinylated or haptenylated tracer is used in live cells, detection requires cell �xation and permeabilization to allow access to �uorescent dye– or enzyme-labeled conjugates of avidin and streptavidin (Section 7.6) or of antibodies (Section 7.4).

In many of these tracing applications, the physical dimensions of the tracer molecule are an important consideration. We o�er �uores-cent tracers ranging in size from small molecules about 1 nm in diam-eter to polystyrene microspheres up to 15 µm in diameter. �is chapter discusses our diverse selection of �uorescent tracers, as well as biotin derivatives and other tracers:

• Cell-permeant cytoplasmic labels (Section 14.2). We have de-veloped several thiol-reactive CellTracker™ probes (Figure 14.1.1), which yield �uorescent products that are retained in many live

Table 14.1 Techniques for loading molecules into the cytoplasm.

Method of Plasma Membrane Breach Size of Molecules Loaded (MW) *

Chemical

ATP 1 1000

Cell-penetrating peptides 2–5 (CPP) 150,000; (DNA)

In�ux™ pinocytic cell-loading reagent 6–8 (I14402) 150,000

α-Toxin of Staphylococcus aureus 9,10 1000

Vehicle

Red blood cell fusion 11,12 300,000

Cationic lipids 13–15 (Lipofectamine® reagents) (DNA)

Mechanical

Microinjection 16,17 150,000

Whole-cell patch clamping 18 Very high

Carbon nanotubes and nanowires 19–21 150,000; (DNA)

Scrape loading 8 500,000; (DNA)

Ultrasonication 22 70,000; (DNA)

Microprojectile bombardment 23,24 10,000; (DNA)

Glass beads 25 150,000

Electrical

Electroporation 26–29 † 150,000; (DNA)

* Molecular weight (MW) of largest molecules reported loaded (DNA is listed separately if it has been successfully introduced by a technique). † We recommend the Neon™ Transfection System (www.invitrogen.com/handbook/neon) for e�cient electroporation of cultured cell lines, primary cells and stem cells.1. J Neurosci (2003) 23:4054; 2. Chembiochem (2010) 11:325; 3. Bioconjug Chem (2009) 20:249; 4. Methods Mol Biol (2009) 480:101; 5. Org Biomol Chem (2008) 6:4516; 6. Methods Enzymol (2006) 414:211; 7. Infect Immun (2006) 74:3673; 8. Antisense Nucleic Acid Drug Dev (2000) 10:263; 9. Proc Natl Acad Sci U S A (2001) 98:3185; 10. Methods (2003) 30:191; 11. Cell (1975) 5:371; 12. J Cell Biol (1985) 101:19; 13. J Biol Chem (2007) 282:22953; 14. Mol Ther (2004) 9:443; 15. Biotechniques (2000) 28:510; 16. Dev Cell (2007) 13:523; 17. J Biomed Sci (2003) 10:328; 18. Neuron (2001) 30:737; 19. Nano Lett (2009) 9:2193; 20. Proc Natl Acad Sci U S A (2010) 107:1870; 21. Proc Natl Acad Sci U S A (2007) 104:8218; 22. BMC Cancer (2002) 2:20; 23. J Neurosci Methods (2009) 184:332; 24. J Neurosci Methods (2005) 141:41; 25. J Cell Sci (1987) 88:669; 26. PLoS One (2009) 4:e7966; 27. Nat Protoc (2009) 4:862; 28. J Neurosci Meth (2009) 177:273; 29. J Neurosci Methods (2009) 178:80.

cells through several generations and are not transferred to adja-cent cells in a population, except possibly by transport through gap junctions. �ese probes represent a signi�cant breakthrough in the cellular retention of �uorescent dyes and are ideal long-term tracers for transplanted cells or tissues.

• Microinjectable cytoplasmic labels (Section 14.3). Polar trac-ers such as lucifer yellow CH, Cascade Blue® hydrazide, the Alexa Fluor® hydrazides and biocytin are membrane-impermeant probes that are usually introduced into cells by whole-cell patch clamp-ing, iontophoresis, osmotic lysis of pinocytic vesicles or comparable methods 2,3 (Table 14.1). �ese tracers are commonly used to investi-gate cell–cell and cell–liposome fusion, as well as membrane perme-ability 4 and transport through gap junctions 5 or cell uptake during pinocytosis (Section 16.1).

• Nissl stains for retrograde tracing in neurons (Section 14.3). We have developed �ve �uorescent Nissl stains that not only provide a wide spectrum of �uorescent colors for staining neurons, but are also more sensitive than the conventional dyes.




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Section 14.1 Choosing a Tracer

REFERENCES1. PLoS Comput Biol (2009) 5:e1000334; 2. J Neurosci Methods (2002) 117:159; 3. Nat Protoc (2009) 4:862; 4. Cytometry (1995) 21:230; 5. Biotechniques (2008) 45:33.

• Fluorescent microspheres (Section 14.6). Molecular Probes® FluoSpheres® and TransFluoSpheres® �uorescent microspheres—which contain ~102 to ~1010 �uorescent dyes per bead are intensely �uorescent tracers (Figure 14.1.3). Although other multiply labeled particles such as our BioParticles® �uorescent bacteria (Section 16.1) may be used as tracers, they are o�en not biologically inert nor are they as physically durable as �uorescent microspheres. �ese properties make �uorescent beads particularly useful as long-term markers for transplantation studies. Submicron microspheres can be injected into cells or taken up by phagocytosis. Much larger (10–15 µm) beads provide an alternative to radioactive microspheres for determination of organ blood �ow, and intermediate-sized (1–5 µm) microspheres are useful for studies that trace inhaled particles.

• Qdot® nanocrystal tracers (Section 14.6). Qtracker® Cell Labeling Kits provide spectrally distinct Qdot® nanocrystals that have been functionalized on their surface with polyarginine peptides to facili-tate spontaneous uptake by live cells. Qtracker® non-targeted quan-tum dots are designed for small animal in vivo imaging, and espe-cially for studying vascular structure a�er microinjection. �ese nanocrystals exhibit intense red or near-infrared �uorescence emission for maximum transmission through tissues and avoid-ance of background auto�uorescence

• Proteins and protein conjugates (Section 14.7). Our �uorescent protein tracers have molecular weights between ~12,000 (chol-era toxin subunit B conjugates) and ~240,000 daltons (B- and R-phycoerythrin). �eir applications are sometimes similar to those of the �uorescent dextrans; however, unlike the polydisperse dextrans, �uorescent protein tracers have molecular weights that are reasonably well de�ned.

Figure 14.1.3 A photomicrograph of a multicolor mixture of Molecular Probes® FluoSpheres® �uorescent microspheres overlaid with a di�erential interference contrast (DIC) image of the same �eld. Molecular Probes® proprietary �uorescent dye technology is used to produce a range of intensely �uorescent FluoSpheres® microspheres labeled with biotin, streptavidin and NeutrAvidin™ biotin–binding protein, providing important tools for improving the sensi-tivity of �ow cytometry applications and immunodiagnostic assays.

Figure 14.1.2 The tracer DiI (D282, D3911) used as a diagnostic tool to evaluate retinal and tectal patterns in a chimeric 5-day-old zebra�sh brain. Acerebellar (Fgf8) gene expression is required for development of midbrain polarity and correct retinotopic mapping of retinal ganglion cell axons. One eye of this brain was wild type, whereas the other was mutant for fgf8 (derived from homozygous acerebellar donors). The chimera was obtained by trans-planting eye primordia, and the eyes were subsequently removed for photography. Retinal ganglion cell axons in the dorsonasal retina were labeled with DiI and nuclei were counter-stained with SYTOX® Green nucleic acid stain (S7020). Axons from the wild-type eye termi-nate correctly in the posterior tectum only, whereas axons from the mutant eye terminate inappropriately in the anterior and posterior tectum. This digital image, previously published on the cover of Development 125 (13) (1999), was created by Alexander Picker and Michael Brand, Neurobiology, University of Heidelberg. The image is used with the permission of The Company of Biologists, Ltd.

• Membrane tracers—DiI, DiO, DiD, DiR, DiA, R18, FM® 1-43, FM® 4-64 and their analogs (Section 14.4). Lipophilic carbocya-nine, aminostyryl and rhodamine dyes can be introduced into membranes by direct application of a dye crystal onto a cell, by bulk loading from aqueous dispersions prepared from our Vybrant® DiI, DiO and DiD cell-labeling solutions or by application of the NeuroTrace® DiI, DiO and DiD tissue-labeling pastes. Lateral dif-fusion of the dye within the membrane eventually stains the entire cell. �ese probes are widely used for neuroanatomical tracing and long-term assays of cell–cell association. Some of our DiI and DiO analogs exhibit superior solubility and brightness and, in some cases, produce a cell-staining pattern that persists through �xation by aldehyde-based reagents and through acetone permeabilization (Figure 14.1.2).

• Fluorescent and biotinylated dextran conjugates (Section 14.5). Dextran conjugates are ideal cell-lineage tracers because they are relatively inert, exhibit low toxicity and are retained in cells for long periods. �ese membrane-impermeant probes are usually loaded into cells by invasive techniques such as micro-injection, whole-cell patch clamping, scrape loading, micropro-jectile bombardment, electroporation or osmotic shock (Table 14.1). Availability of dextrans in a range of molecular weights makes them useful as size-exclusion probes for determining pore sizes in membranes.



595




Section 14.2 Membrane-Permeant Reactive Tracers

14.2 Membrane-Permeant Reactive TracersThiol-Reactive CellTracker™ Probes

Molecular Probes® CellTracker™ reagents are �uorescent chloromethyl derivatives that freely di�use through the membranes of live cells (Figure 14.2.1, Figure 14.2.2, Figure 14.2.3). Once inside the cell, these mildly thiol-reactive probes undergo what is believed to be a glutathione S-transferase–mediated reaction to produce membrane-impermeant glutathione–�uorescent dye adducts, al-though our experiments suggest that they may also react with other intracellular components (Figure 14.2.4). Regardless of the mechanism, many cell types loaded with the CellTracker™ probes are both �uorescent and viable for at least 24 hours a�er loading and o�en through several cell divi-sions. Most other cell-permeant �uorescent dyes, including the acetoxymethyl (AM) esters of calcein and BCECF (Section 15.2), are retained in viable cells for no more than a few hours at physiological temperatures (Figure 14.2.5). Furthermore, unlike the free dye, the peptide–�uorescent dye adducts contain amino groups and can therefore be covalently linked to surrounding biomolecules by �xa-tion with formaldehyde or glutaraldehyde. �is property permits long-term storage of the labeled cells or tissue and, in cases where the cognate anti-dye antibody is available (see below), ampli�cation of the dye hapten by standard immunohistochemical techniques. Fixation without loss of the tracer also facilitates the safe handling and analysis of cells containing pathogens.

Figure 14.2.1 Detection of organisms in marine sediments by incubating an intact sediment core sample with the �xable, cell-permeant CellTracker™ Green CMFDA (C2925, C7025). The core sample was subsequently embedded, sectioned and ex-amined for �uorescently labeled organisms. The micrograph reveals the microorganism Leptohalysis scotti, a marine ben-thic foraminifera. Image contributed by Joan M. Bernhard, Wadsworth Center, New York State Department of Health.

Figure 14.2.2 HL60 cells that have been stained with CellTracker™ Orange CMTMR (C2927) and then mixed with WEHI 7.1 cells stained with CellTracker™ Green CMFDA (C2925, C7025) (left). Several minutes after initiating cell–cell electrofusion, a CMTMR-stained HL60 cell is observed fusing with a CMFDA-stained WEHI 7.1 cell; cytoplasmic mixing is evident by the ap-pearance of yellow �uorescence. After electrofusion is complete, dual-�uorescing (yellow) hybrids can be easily distinguished (right). Images contributed by Mark J. Jaroszeski, University of South Florida.

Figure 14.2.3 Individual populations of mouse myeloma (P3X) cells stained with our reactive tracers—CellTracker™ Orange CMTMR (C2927), CellTracker™ Green CMFDA (C2925) and CellTracker™ Blue CMAC (C2110). Each of three cell populations was stained with a di�erent tracer and then the populations were mixed, demonstrating that these trac-ers allow simultaneous long-term monitoring of di�erent groups of cells in transplantation and other assays.

Figure 14.2.4 Intracellular reactions of our �xable CellTracker™ Green CMFDA (5-chloromethyl�uorescein diacetate; C2925, C7025). Once this membrane-permeant probe enters a cell, esterase hydrolysis converts non�uorescent CMFDA to �uores-cent 5-chloromethyl�uorescein, which can then react with thiols on proteins and peptides to form aldehyde-�xable conju-gates. This probe may also react with intracellular thiol-containing biomolecules �rst, but the conjugate is non�uorescent until its acetates are removed.

OO

CH2CI

O O

OCCH3CH3CO O

CH2CI

COH

O

HO O

CH2S

COH

O

HO

Peptide

O

Esterases SH Peptide

O O

Figure 14.2.5 Loading and retention characteristics of intracellular marker dyes. Cells of a human lymphoid line (GePa) were load-ed with the following cell-permeant acetoxymethyl ester (AM) or acetate derivatives of �uorescein: 1) calcein AM (C1430, C3099, C3100MP), 2) BCECF AM (B1150), 3) �uorescein diacetate (FDA, F1303), 4) carboxy�uorescein diacetate (CFDA, C1354) and 5) CellTracker™ Green CMFDA (5-chloromethyl�uorescein diacetate, C2925, C7025). Cells were incubated in 4 µM staining solutions in Dulbecco's modi�ed eagle medium containing 10% fetal bovine serum (DMEM+) at 37°C. After incubation for 30 minutes, cell sam-ples were immediately analyzed by �ow cytometry to determine the average �uorescence per cell at time zero (0 hours). Retained cell samples were subsequently washed twice by centrifugation, resuspended in DMEM+, maintained at 37°C for 2 hours and then analyzed by �ow cytometry. The decrease in the average �uorescence intensity per cell in these samples relative to the time zero samples indicates the extent of intracellular dye leakage during the 2-hour incubation period.

Rel

ativ

e ce

llula

r �u

ores

cenc

e

Intracellular marker

104

103

102

101

1001 2 3 4 5

0 hours2 hours




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CellTracker™ Probes in a Variety of Fluorescent ColorsMolecular Probes® CellTracker™ product line includes reactive chloromethyl derivatives of:

• Blue-�uorescent 7-aminocoumarin (CellTracker™ Blue CMAC, C2110, Figure 14.2.6)• Blue-�uorescent 7-hydroxycoumarin (CellTracker™ Blue CMHC, C2111, Figure 14.2.7)• Blue-�uorescent 6,8-di�uoro-7-hydroxycoumarin (CellTracker™ Blue CMF2HC, C12881,

Figure 14.2.8)• Violet-�uorescent 2,3,6,7-tetrahydro-9- bromomethyl-1H,5H-quinolizino(9,1-gh)coumarin

(CellTracker™ Violet BMQC, C10094, Figure 14.2.9)• Green-�uorescent �uorescein diacetate (CellTracker™ Green CMFDA, C2925 and C7025,

Figure 14.2.4)• Green-�uorescent BODIPY® derivative (CellTracker™ Green BODIPY®, C2102, Figure 14.2.10)• Orange-�uorescent tetramethylrhodamine (CellTracker™ Orange CMTMR, C2927, Figure

14.2.11)• Orange-�uorescent CellTracker™ Orange CMRA (C34551, Figure 14.2.12)• Red-�uorescent CellTracker™ Red CMTPX (C34552, Figure 14.2.13)

CellTracker™ Green CMFDA freely di�uses into the cell, where cytosolic esterases cleave the acetate groups, releasing the �uorescent product; CellTracker™ Orange CMRA also requires es-terase cleavage to activate its �uorescence. �e CellTracker™ Blue CMAC, CMHC and CMF2HC, CellTracker™ Violet BMQC, CellTracker™ Green BODIPY®, CellTracker™ Orange CMTMR and CellTracker™ Red CMTPX probes do not require enzymatic cleavage to activate their �uores-cence. CellTracker™ Orange CMRA is a rhodol-based �uorophore with an overall net charge at neutral pH that is expected to be negative, in contrast with the overall net positive charge of the tetramethylrhodamine-based CellTracker™ Orange CMTMR. �us, unlike CellTracker™ Orange CMTMR, CellTracker™ Orange CMRA should remain primarily in the cytoplasm instead of being sequestered inside actively respiring mitochondria. �e long-wavelength CellTracker™ Red CMTPX exhibits bright red �uorescence that is easily distinguished from that of blue-, green- and far-red–�uorescent probes, including CellTracker™ Green CMFDA and CFSE 1–3 (see below). We also o�er the green-�uorescent BODIPY® 493/503 methyl bromide (B2103, Figure 14.2.14), which has slightly greater thiol-reactivity than do chloromethyl derivatives.

Applications for CellTracker™ Probes�e thiol-reactive CellTracker™ Green CMFDA is suitable for long-term cell labeling and has

been frequently used for in vitro labeling of cells prior to adoptive transfer to animals and tis-sues.2,4,5 Other prominent applications include the analysis of cell–cell fusion,6,7 cell adhesion 8,9 and multidrug resistance transporter function.10,11

CellTracker™ Green CMFDA was used to track wild-type and myosin II mutant Dictyostelium discoideum cells within aggregation streams during early multicellular morphogenesis; di�er-entiation and morphogenesis pathways were reportedly una�ected in labeled cells imaged over several days by confocal laser-scanning microscopy.12,13 CellTracker™ Green BODIPY® was used to label CD4+ T cells and follow their activation and proliferation in mice with the immunode-�ciency syndrome MAIDS.14 CellTracker™ Green CMFDA and some other CellTracker™ dyes have been utilized to selectively label intracellular Chlamydia psittaci bacteria in infected cells.15

CellTracker™ Orange CMTMR has been used to stain the cytoplasm of engrailed-expressing Drosophila cells in an in vitro reconstruction experiment,16 to follow T-cell di�erentiation and maturation 17 and to trace implanted tumor cells for at least two weeks.18,19 Chlamydia labeled

Figure 14.2.6 CellTracker™ Blue CMAC (7-amino-4-chloro-methylcoumarin, C2110).

Figure 14.2.7 CellTracker™ Blue CMHC (4-chloromethyl-7-hydroxycoumarin, C2111).

Figure 14.2.8 CellTracker™ Blue CMF2HC (4-chloromethyl-6,8-di�uoro-7-hydroxycoumarin; C12881).

O

CH2Br

N O

Figure 14.2.9 CellTracker™ Violet BMQC (2,3,6,7-tetrahy-dro-9-bromomethyl-1H,5H-quinolizino(9,1-gh)coumarin; C10094).

Figure 14.2.10 CellTracker™ Green BODIPY® (8-chloro-methyl-4,4-di�uoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene; C2102).

Figure 14.2.11 CellTracker™ Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; C2927). Figure 14.2.12 CellTracker™ Orange CMRA (C34551).

5

O O

OO

C CH3

O

Cl

N

CH3H3C

H3C

CH3

6

ClCH2

Figure 14.2.13 CellTracker™ Red CMTPX (C34552).

CClCH2O

N O N

CH3

CH3CH3CH3H3C

H3C CH3

CO

O

CH3

6

5

NH

Figure 14.2.14 BODIPY® 493/503 methyl bromide (8-bro-momethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene; B2103).

NB

N

FF CH3H3C

H3CCH2Br CH3



597





with CellTracker™ Orange CMTMR continued to grow and di�erentiate, and labeled chlamydia isolated from infected cells remained infectious.15

Metabolic activity and drug-induced cytotoxicity were measured with CellTracker™ Blue CMAC in a �uorescence-based microplate assay.20 Peptidase substrates derived from CellTracker™ Blue CMAC yield blue-�uorescent products that are well retained in live cells 21 (Section 10.4). Our CellTracker™ Blue CMF2HC (C12881) has a low pKa that helps ensure that the dye’s conjugates will be ionized and have bright blue �uorescence in the cytoplasm.1,22 CellTracker™ Blue CMAC was used in combination with calcein AM (C1430, C3099, C3100MP; Section 15.6) and the lipophilic tracer DiI (D282, D3911, V22885; Section 14.4) to identify lipid mixing and cytoplasm mixing between labeled e�ector cells and labeled target cells.23

�e ability to �x the intracellular products of CellTracker™ Green CMFDA, CellTracker™ Green BODIPY® and CellTracker™ Orange CMTMR in permeabilized cells permits the stained cells to be probed with our anti–�uorescein/Oregon Green®, anti–BODIPY® FL and anti-tetra-methylrhodamine antibodies, respectively (Section 7.4, Table 7.8).

Other Thiol-Reactive TracersChloromethyl Derivatives of SNARF®-1 and H2DCFDA

Although they were designed for other purposes, the chloromethyl derivatives of our SNARF®-1 pH indicator (C6826, Figure 14.2.15) and of 2 ,ʹ7ʹ-dichlorodihydro�uorescein diac-etate, acetate ester (CM-H2DCFDA, C6827, Figure 14.2.16) possess some unique properties for tracking cells. As with the CellTracker™ probes, cytoplasmic enzymes hydrolytically remove the acetate groups from the membrane-permeant probes, and the chloromethyl moieties become conjugated to intracellular thiols.24 With its long Stokes shi� (Figure 14.2.17), the SNARF®-1 derivative has easily distinguished, red-orange �uorescence in the cytoplasm when excited at the same wavelengths used for the green-�uorescent CellTracker™ Green CMFDA (Figure 14.2.18).

As with other dihydro�uorescein derivatives (Section 18.2), CM-H2DCFDA requires an ad-ditional oxidation step before becoming �uorescent. �is probe is useful for following stimula-tion of oxidative activity by external agents or natural killer (NK) cells over extended periods, as well as for passively labeling cells that lack appropriate oxidative activity and then following their ingestion by scavengers such as neutrophils. CM-H2DCFDA has been used to measure intracel-lular reactive oxygen species (ROS) in cardiac myocytes 25 and in human embryonic kidney 293 (HEK 293) cells stably transfected with the human vanilloid receptor 1 (VR1) cation channel.26

Bimanes: Blue-Fluorescent Reactive Tracers�e bimane derivatives, monobromobimane (mBBr; M1378, M20381) and monochlorobi-

mane (mBCl, M1381MP), are important thiol-derivatization reagents (Section 2.3). �e essen-tially non�uorescent mBBr and mBCl dyes are known to passively di�use across the plasma membrane into the cytoplasm, where they form blue-�uorescent adducts with intracellular glu-tathione and thiol-containing proteins 27 (Section 15.6).

Amine-Reactive TracersCFSE and Its Derivatives

Carboxy�uorescein diacetate succinimidyl ester (5(6)-CFDA, SE; C1157) is commonly re-ferred to as CFSE. Among its many applications, the two most prominent are ex vivo labeling of cells for adoptive transfer and �ow cytometric tracking of cell division by label partition analysis. CFSE is colorless and non�uorescent until its acetate groups are cleaved by intracellular esterases to yield highly �uorescent, amine-reactive 5(6)-carboxy�uorescein succinimidyl ester (equivalent to C1311, Section 1.5). Upon reaction with amine-containing residues of intracellular proteins, the resulting dye–protein adducts are well retained in cells as they move and divide during embryonic development.28,29 In addition, these adducts survive subsequent �xation with formaldehyde or glutaraldehyde. Because it is intrinsically more reactive, the succinimidyl ester of CFDA is more likely to react at sites on the extracellular surface than is CMFDA.

Once incorporated into cells, CFSE remains there—even through cell division—thus pre-venting transfer to unlabeled cells.30 Labeled cells can subsequently be immunohistochemically

Figure 14.2.15 5-(and-6)-Chloromethyl SNARF®-1, acetate (C6826).

(CH3)2N O

OO

OCCH3

O

ClH2C

5

6

Figure 14.2.16 5-(and-6)-Chloromethyl-2’,7’-dichlorodihydro-�uorescein diacetate, acetyl ester (CM-H2DCFDA, C6827).

CH3CO O

COCCH3

OCCH3

O

ClH2C

5

6

H

O

ClCl

OO

Figure 14.2.17 Absorption and �uorescence emission (ex-cited at 488 nm) spectra of carboxy SNARF®-1 in pH 9.0 and pH 6.0 bu�ers.

Figure 14.2.18 Human neutrophils loaded with 5-(and-6)-chloromethyl SNARF®-1 acetate (C6826).




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detected post-mortem using our anti–�uorescein/Oregon Green® antibodies 31–33 (Section 7.4). �e feasibility of using cell-permeant �uorescent tracers to follow asynchronous cell division of natural killer (NK) cells, B cells, T cells, thymocytes, lymphocytes, �broblasts and hematopoietic cells has been demonstrated with CFSE 34–37 (Figure 14.2.19). Lymphocytes labeled with CFSE were detected up to eight weeks a�er injection into mice during lymphocyte migration studies,38–40 and similarly labeled hepatocytes were easily located by �uorescence microscopy even 20 days a�er intrahepatic transplantation.41 CFSE has also been successfully employed to quantitate adhesion of neutro-phils 42 and leukocytes,43 to assay T-cadherin–mediated cell aggregation,44 to follow neurite growth in an in vitro bioassay 45 and to trace fetal cells in culture.46 Di�erent loading concentrations of CFSE can be used to produce cells that can be distinguished by their relative brightness.47 CFSE has been utilized for tracing the transport of viable bacterial cells in groundwater under no-growth conditions for periods of at least 28 days.48 CFSE is also available conveniently packaged for cell-tracing applications in our Vybrant® CFDA SE Cell Tracer Kit (V12883) and for cell proliferation studies in our CellTrace™ CFSE Cell Proliferation Kit (C34554, Figure 14.2.20). �e �uorescent CFSE product has excitation/emission maxima of ~492/517 nm and can be detected using a �uo-rescence microscope, �ow cytometer or �uorescence microplate reader.

Carboxyeosin diacetate succinimidyl ester (C22803) has applications that are expected to be similar to CFSE; however, its �uorescence can be excited and detected at longer wavelengths, possibly permitting two-color tracing experiments of mixed-cell populations. Eosin derivatives are also e�ective reagents for photoablation of cells. Unlike CFSE, �uorescence of the intracel-lular hydrolysis products of the succinimidyl ester of 5-(and 6-)carboxy-2 ,ʹ7ʹ-dichloro�uorescein diacetate (C1165) is relatively insensitive to �uctuations in pH. �is amine-reactive tracer was reported to be more useful than the lipophilic marker DiI (D282, Section 14.4) in an investigation of palatal fusion in rodent embryos.49

CellTrace™ Violet Cell Proliferation KitCellTrace™ Violet stain is an esterase-activated phenolic �uorophore with a succinimidyl ester

substituent for coupling to cell surface and intracellular amines. It is functionally analogous to CFSE, equally partitioning between daughter cells during division resulting in successive 2-fold reductions in cell-associated �uorescence intensity. When analyzed by �ow cytometry, this pro-gressive label partitioning provides a direct indication of cell proliferation status (Figure 14.2.21). In contrast to CFSE, CellTrace™ Violet stain is optimally excited by 405 nm violet diode lasers and generates blue �uorescence (emission peak ~455 nm). Consequently, it can be used in combina-tion with CFSE to track cells from di�erent origins a�er mixing or to analyze proliferation of GFP-expressing cells. �e CellTrace™ Violet Cell Proliferation Kit (C34557) includes the CellTrace™ Violet stain together with dimethylsulfoxide (DMSO) for preparation of a stock solution.

CellTrace™ Oregon Green® 488 Carboxylic Acid Diacetate Succinimidyl Ester�e succinimidyl ester of Oregon Green® 488 carboxylic acid diacetate (carboxy-DFFDA SE)

o�ers several important advantages over CFSE as a �uorescent cell tracer. �is Oregon Green® 488 probe passively di�uses into cells, where it is colorless and non�uorescent until its acetate groups are removed by intracellular esterases to yield a highly green-�uorescent, amine-reactive dye. Upon reaction with intracellular amines, the probe forms Oregon Green® 488 conjugates that are well-retained by cells. Unlike �uorescein derivatives, however, Oregon Green® 488 de-rivatives exhibit bright green �uorescence that is not pH dependent at typical cellular pH val-ues. Moreover, Oregon Green® 488 probes are usually brighter and more photostable than �uores-cein probes. We o�er carboxy-DFFDA SE in a 1 mg unit size (O34550) and specially packaged in a set of 20 vials, each containing 50 µg (CellTrace™ Oregon Green® 488 carboxylic acid diacetate succinimidyl ester, C34555).

CellTrace™ Far Red DDAO-SECellTrace™ Far Red DDAO-SE (C34553) is a �xable, far-red–�uorescent tracer for long-term

cell labeling. �e succinimidyl ester (SE) reactive group forms a strong covalent attachment to primary amines that occur in proteins and other biomolecules inside and outside of cells. With its far-red �uorescence, CellTrace™ Far Red DDAO-SE has minimal spectral overlap with most other �uorophores (Figure 14.2.22) and thus can be used simultaneously with almost any blue, green or orange �uorophores including Green Fluorescent Protein 50–52 (GFP).

Figure 14.2.19 Tracking of asynchronous cell division us-ing 5-(and 6-)carboxy�uorescein diacetate, succinimidyl ester (5(6)-CFDA SE or CFSE; C1157; V12883) labeling and �ow cytometry. Cell division results in sequential halving of the initial �uorescence, resulting in a multipartite cellular �uorescence histogram. The peaks labeled 0, 1, 2, 3, 4 and 5 represent successive generations.

10 100 1000 10000

Cellular �uorescence

Num

ber

of c

ells

cou

nted

5

4

3

21

0

Figure 14.2.20 Following cell proliferation in human pe-ripheral blood lymphocytes using the CellTrace™ CFSE Cell Proliferation Kit C34554). Human peripheral blood lympho-cytes were harvested and stained with CellTrace™ CFSE (car-boxy�uorescein diacetate, succinimidyl ester; 5(6)-CFDA, SE) on Day 0. A portion of the population was arrested at the parent generation using mitomycin C (red peak). The remainder of the sample was stimulated with phytohemag-glutinin and allowed to proliferate for 5 days. Solid green peaks represent successive generations.

CellTrace™ CFSE �uorescence

Num

ber

of c

ells

cou

nted

100 101 102 103 104

Figure 14.2.21 Human peripheral blood lymphocytes were harvested and stained with CellTrace™ Violet stain (C34557). The violet peaks represent successive genera-tions of cells stimulated with mouse anti–human CD3 and Interleukin-2 and grown in culture for 7 days. The peak out-lined in black represents cells that were grown in culture for 7 days with no stimulus.

CellTrace™ Violet �uorescence

Num

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of c

ells

cou

nted

100 101 102 103 105104


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

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Amine-Reactive SNARF®-1 Carboxylic Acid AcetateTo permit simultaneous long-term tracing of mixed-cell populations using di�erent �uores-

cent colors, we have developed an amine-reactive probe whose applications are similar to those of CFSE.53 Cells labeled with our succinimidyl ester of SNARF®-1 carboxylic acid acetate (S22801, Figure 14.2.23) have a red-orange �uorescence that can easily be distinguished from that of cells loaded with green-�uorescent tracers such as CFSE. However, the �uorescence intensity of cells loaded with this SNARF®-1 derivative will not be as high as that of cells loaded with the same con-centration of CFSE. �us, it is necessary to adjust the reagent concentration and/or select optical �lters to appropriately balance the �uorescence intensities when doing two-color experiments.

Figure 14.2.22 Normalized absorption and �uorescence emission spectra of DDAO.

Ab

sorp

tion

Wavelength (nm)

Fluo

resc

ence

em

issi

on

400 500 600 750 800650 700550450

Figure 14.2.23 SNARF®-1 carboxylic acid, acetate, succin-imidyl ester (S22801).

REFERENCES1. Nat Immunol (2008) 9:155; 2. J Immunol (2008) 181:3947; 3. Cancer Res (2008) 68:6341; 4. J Neurosci (2008) 28:5965; 5. Nat Med (2008) 14:213; 6. Nat Methods (2009) 6:147; 7. Proc Natl Acad Sci U S A (2003) 100:2397; 8. Anal Chem (2008) 80:7543; 9. Nature (2008) 451:465; 10. Eur J Pharm Biopharm (2008) 69:396; 11. Mol Pharm (2008) 5:787; 12. Dev Biol (1995) 170:434; 13. J Cell Sci (1995) 108:1105; 14. J Immunol (2002) 169:722; 15. J Microbiol Methods (2000) 40:265; 16. Nature (1993) 363:549; 17. Stem Cells (1996) 14:132; 18. Anticancer Res (1995) 15:719; 19. Prostate (1998) 35:1; 20. Clin Chem (1995) 41:1906; 21. J Biol Chem (1993) 268:23593; 22. Science (2009) 323:1743; 23. J Cell Biol (1998) 140:315; 24. Cytometry A (2007) 71:709; 25. J Biol Chem (1999) 274:19323; 26. J Pharmacol Exp �er (2002) 300:9; 27. Glia (2004) 45:59; 28. J Cell Biol (1985) 101:610; 29. J Cell Biol (1986) 103:2649; 30. J Cell Sci (1992) 102:789; 31. Nat Biotechnol (2008) 26:215; 32. Am J Pathol (2006) 169:2223; 33. Development (1998) 125:201; 34. Methods Mol Biol (2009) 510:415; 35. Clin Chem Lab Med (2005) 43:841; 36. Nat Protoc (2007) 2:2049; 37. Nat Protoc (2007) 2:2057; 38. J Immunol Methods (2004) 286:69; 39. J Immunol Methods (1990) 133:87; 40. Cytometry (1992) 13:739; 41. Transplant Proc (1992) 24:2820; 42. J Immunol Methods (1994) 172:25; 43. J Immunol Methods (1994) 172:115; 44. J Cell Biol (1992) 119:451; 45. J Neurosci Methods (1991) 39:193; 46. J Neurosci Methods (1992) 44:7; 47. J Exp Med (1996) 183:2313; 48. Appl Environ Microbiol (2000) 66:4486; 49. Development (1992) 116:1087; 50. Cytometry A (2009) 75:440; 51. J Immunol (2008) 181:4043; 52. J Immunol (2007) 179:6808; 53. Cytometry B Clin Cytom (2007) 72:458.

DATA TABLE 14.2 MEMBRANE-PERMEANT REACTIVE TRACERSCat. No. MW Storage Soluble Abs EC Em Solvent NotesB2103 341.00 F,D,L DMSO, MeCN 533 62,000 561 CHCl3 1C1157 557.47 F,D DMF, DMSO

600




Section 14.3 Polar Tracers

PRODUCT LIST 14.2 MEMBRANE-PERMEANT REACTIVE TRACERSCat. No. Product QuantityB2103 BODIPY® 493/503 methyl bromide (8-bromomethyl-4,4-di�uoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) 5 mgC1165 CDCFDA, SE (5-(and-6)-carboxy-2’,7’-dichloro�uorescein diacetate, succinimidyl ester) *mixed isomers* 25 mgC22803 CEDA, SE (5-(and-6)-carboxyeosin diacetate, succinimidyl ester) *mixed isomers* 5 mgC34554 CellTrace™ CFSE Cell Proliferation Kit *for �ow cytometry* 1 kitC34553 CellTrace™ Far Red DDAO-SE *special packaging* 20 x 50 µgC34555 CellTrace™ Oregon Green® 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA, SE) *cell permeant* *mixed isomers* 20 x 50 µgC34557 CellTrace™ Violet Cell Proliferation Kit *for �ow cytometry* 1 kitC2110 CellTracker™ Blue CMAC (7-amino-4-chloromethylcoumarin) 5 mgC12881 CellTracker™ Blue CMF2HC (4-chloromethyl-6,8-di�uoro-7-hydroxycoumarin) 5 mgC2111 CellTracker™ Blue CMHC (4-chloromethyl-7-hydroxycoumarin) 5 mgC2102 CellTracker™ Green BODIPY® (8-chloromethyl-4,4-di�uoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene) 5 mgC2925 CellTracker™ Green CMFDA (5-chloromethyl�uorescein diacetate) 1 mgC7025 CellTracker™ Green CMFDA (5-chloromethyl�uorescein diacetate) *special packaging* 20 x 50 µgC34551 CellTracker™ Orange CMRA *special packaging* 20 x 50 µgC2927 CellTracker™ Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine) *mixed isomers* 1 mgC34552 CellTracker™ Red CMTPX *special packaging* 20 x 50 µgC10094 CellTracker™ Violet BMQC (2,3,6,7-tetrahydro-9-bromomethyl-1H,5H-quinolizino(9,1-gh)coumarin) 5 x 100 µgC1157 5(6)-CFDA, SE; CFSE (5-(and-6)-carboxy�uorescein diacetate, succinimidyl ester) *mixed isomers* 25 mgC6827 5-(and-6)-chloromethyl-2’,7’-dichlorodihydro�uorescein diacetate, acetyl ester (CM-H2DCFDA) *mixed isomers* *special packaging* 20 x 50 µgC6826 5-(and-6)-chloromethyl SNARF®-1, acetate *mixed isomers* *special packaging* 20 x 50 µgM1378 monobromobimane (mBBr) 25 mgM20381 monobromobimane (mBBr) *FluoroPure™ grade* 25 mgM1381MP monochlorobimane (mBCl) 25 mgO34550 Oregon Green® 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA, SE) *mixed isomers* 1 mgS22801 SNARF®-1 carboxylic acid, acetate, succinimidyl ester *special packaging* 10 x 50 µgV12883 Vybrant® CFDA SE Cell Tracer Kit 1 kit

14.3 Polar TracersFixable Polar Tracers

We prepare a wide variety of highly water-soluble dyes and other detectable probes that can be used as cell tracers. In most cases, the polarity of these water-soluble probes is too high to permit them to passively di�use through cell membranes. Consequently, special methods for loading the dyes into cells must be employed, including microinjection, pinocytosis or techniques that temporarily permeabilize the cell’s membrane 1,2 (Table 14.1). Our In�ux™ pinocytic cell-loading reagent (I14402, see below and Section 19.8) is particularly useful for loading many of the polar tracers in this section—as well as the dextrans and �uorescent proteins described in Section 14.5 and Section 14.7—into many types of cells. Permeabilization of cells with staphylococcal α-toxin 3 or the saponin ester β-escin is reported to make the membrane of smooth muscle cells perme-able to low molecular weight (

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Figure 14.3.2 Confocal image stack of a 10,000 MW Calcium Green™ dextran–labeled (C3713) climbing �ber in a sagit-tal cerebellar slice, showing incoming axon and terminal arborization (in yellow). The Purkinje cell innervated by this climbing �ber was labeled with Alexa Fluor® 568 hydrazide (A10437, A10441) via a patch pipette and visually identi�ed using bright-�eld microscopy. Image contributed by Anatol Kreitzer, Department of Neurobiology, Harvard Medical School.

and Alexa Fluor® 568 hydrazides (A20501MP, A10437, A10441; Figure 14.3.2), red-�uorescent Alexa Fluor® 594 hydrazide 10 (A10438, A10442) and far-red–�uorescent Alexa Fluor® 633 hy-drazide, Alexa Fluor® 647 hydrazide and Alexa Fluor® 647 hydroxylamine (A30634, A20502, A30632) are likely among the best overall polar tracers in each of their various spectral rang-es.11 �ese low molecular weight, cell membrane–impermeant molecules (Alexa Fluor® 350 hydrazide, 349 daltons; Alexa Fluor® 350 hydroxylamine, 585 daltons; ~570–760 daltons for the Alexa Fluor® 488, 568 and 594 hydrazides and hydroxylamine; and about 1200 daltons for the Alexa Fluor® 555 and 647 hydrazides and hydroxylamine) possess several properties that are superior to those of the widely used neuronal tracer lucifer yellow CH (L453, L682, L1177, L12926). Like lucifer yellow CH, the hydrazide moiety of the Alexa Fluor® derivatives makes these tracers �xable by common aldehyde-based �xatives. We have determined that Alexa Fluor® 594 hydrazide has a water solubility of ~84 mg/mL and the other Alexa Fluor® hydrazides are likely to have comparable or higher water solubility.

Our rabbit polyclonal antibody to the Alexa Fluor® 488 �uorophore (A11094, Section 7.4) quenches the �uorescence of the Alexa Fluor® 488 dye (Anti–Lucifer Yellow Dye, Anti–Alexa Fluor® 405/Cascade Blue® Dye and Anti–Alexa Fluor® 488 Dye Antibodies—Note 14.1) and, following cell �xation and permeabilization, can be used in conjunction with the reagents in our Tyramide Signal Ampli�cation (TSA™) Kits (Section 6.2) to amplify the signal or with the anti–rabbit IgG conjugate of NANOGOLD® or Alexa Fluor® FluoroNanogold™ 1.4 nm gold clusters (N24916, A24926, A24927; Section 7.2) and the associated LI Silver Enhancement Kit (L24919, Section 7.2) for correlated �uorescence and light microscopy studies.

Although lucifer yellow CH can be used for confocal laser-scanning microscopy, its extinction coe�cient at the 488 nm spectral line of the argon-ion laser (~700 cm–1M–1) is only about 1% of that of Alexa Fluor® 488 hydrazide and Alexa Fluor® 488 hydroxylamine (≥71,000 cm–1M–1) (Figure 14.3.3). Furthermore, the high photostability of the Alexa Fluor® dyes permits their detection in very �ne structures that cannot be seen with lucifer yellow CH staining. All of these Alexa Fluor® dyes are remarkably bright and photostable. In addition, the Alexa Fluor® hydrazide salts have high water solubility (typically greater than 8%). We o�er the Alexa Fluor® 568 and Alexa Fluor® 594 hydrazides either as solids (A10437, A10438) or as 10 mM solutions in 200 mM KCl (A10441, A10442). �e 10 mM solutions have been �ltered through a 0.2 µm �lter to remove any insoluble material prior to packaging. Alexa Fluor® 488 hydrazide, Alexa Fluor® 555 hydrazide, Alexa Fluor® 633 hydrazide and Alexa Fluor® 647 hydrazide (A10436, A20501MP, A30634, A20502) and Alexa Fluor® 350, Alexa Fluor® 488 and Alexa Fluor® 647 hydroxylamines (A30627, A30629, A30632) are only avail-able as solids. Our Alexa Fluor® 350 hydrazide and Alexa Fluor® 350 hydroxylamine, which

Molecular Probes® anti–lucifer yellow dye antibodies were speci�cally developed to overcome certain limitations of lucifer yellow CH (L453, L682, L12926, L1177). Lucifer yellow CH is an aldehyde-�xable �uorescent cell tracer that has long been used by neuroscientists to identify patterns of gap junctional communication,1 to assay the outgrowth of developing neurons 2 and to characterize the morphology of neurons from which electrical record-ings have been made.3 Even though the cell soma of a lucifer yellow CH–�lled neuron may be brightly stained, its �ner processes can sometimes be faint and may fade rapidly or be obscured by the more intensely stained portions of the neuron. Investigators have been able to overcome these limitations by using anti–lucifer yellow dye antibodies in conjunction with standard enzyme-mediated immunohistochemical methods to develop a more permanent, fade-free signal for light microscopy.4–7 Anti–lucifer yellow dye antibodies have also been used to develop tissue for electron microscopy 8 and to dis-tinguish neurons �lled with lucifer yellow CH from those injected with the

lectin Phaseolus vulgaris leucoagglutinin 9 (PHA-L). Molecular Probes® Cascade Blue® hydrazide (C3239), which can also be �xed in place with aldehyde-based �xatives, can potentially be used as a second label with lucifer yellow CH to characterize the morphology of interacting neurons.

For these applications,we o�er unconjugated and biotinylated rabbit polyclonal anti–lucifer yellow dye (A5750, A5751) and anti–Alexa Fluor® 405/Cascade Blue® dye (A5760) antibodies. Similarly, our rabbit polyclonal anti–Alexa Fluor® 488 dye antibody (A11094) can be used to detect Alexa Fluor® 488 hydrazide (A10436), Alexa Fluor® 488 hydroxylamine (A30629), Alexa Fluor® 488 cadaverine (A30676) and Alexa Fluor® 488 biocytin (A12924) in �xed-cell preparations. See Section 7.4 (Table 7.8) for a complete description of these and other anti-�uorophore and anti-biotin antibodies.

1. Biotechniques (2008) 45:33; 2. Science (1988) 242:700; 3. J Neurosci (2003) 23:4700; 4. J Neurosci (1994) 14:5267; 5. J Neurosci Methods (1992) 41:45; 6. J Comp Neurol (1990) 296:598; 7. Dev Biol (1982) 94:391; 8. Circ Res (1992) 70:49; 9. J Neurosci Methods (1990) 33:207.

NOTE 14.1

Anti–Lucifer Yellow Dye, Anti–Alexa Fluor® 405/Cascade Blue® Dye and Anti–Alexa Fluor® 488 Dye Antibodies

Figure 14.3.3 Absorption spectra showing that the molar extinction coe�cient (EC) at 488 nm of Alexa Fluor® 488 hy-drazide (A10436) in water (green line) is approximately 100-fold greater than that of lucifer yellow CH (L453, L682, L1177, L12926) in water (blue line).

488 nm

Ext

inct

ion

coef

�cie

nt (c

m-1

M-1

)Wavelength (nm)

75000

15000

45000

0350 400 450 500 550

30000

60000




602





Figure 14.3.5 Lucifer yellow CH, lithium salt (L453).

Figure 14.3.6 Cultured left-upper quadrant neurons from Aplysia californica that have been microinjected with either lucifer yellow CH (L453, L682, L1177, L12926) or sulforho-damine 101 (S359). These neurons display an extensive array of overlapping processes (J Neurophysiol (1991) 66:316). Image contributed by David Kleinfeld, AT&T Bell Laboratories, and Brian Salzberg, University of Pennsylvania School of Medicine.

Figure 14.3.7 Absorption spectra for equal concentrations of Cascade Blue® hydrazide (C687, C3221, C3239) and lucifer yellow CH (L453, L682, L1177, L12926) in water.

Ab

sorp

tion

Wavelength (nm)250

Cascade Blue®

Lucifer yellow

350 450 550

Figure 14.3.4 Alexa Fluor® 350 hydrazide, sodium salt (A10439).

are sulfonated coumarin derivatives (Figure 14.3.4), are some of the few polar tracers that exhibit bright blue �uorescence.

Other Alexa Fluor® DerivativesTo allow ampli�cation of signals, especially in the �ner processes

of dye-�lled neurons, we also o�er Alexa Fluor® 488 biocytin (A12924), Alexa Fluor® 546 biocytin (A12923) and Alexa Fluor® 594 biocytin (A12922). �ese unique probes combine our Alexa Fluor® 488, Alexa Fluor® 546 and Alexa Fluor® 594 �uorophores with biotin and an alde-hyde-�xable primary amine (see “Fluorescent Biotin Derivatives,” be-low). In addition, we o�er the bright blue-�uorescent Alexa Fluor® 405 cadaverine (A30675, see below) as well as several other Alexa Fluor® ca-daverines (Section 3.3, Table 3.2), all of which should be useful as tracing molecules because they are exceptionally bright, small and water solu-ble, and they each contain an aldehyde-�xable functional group. Alexa Fluor® 546 biocytin has been used to label streptavidin-coated particles in order to quantitate �uorescence signals in an automated imaging sys-tem designed for analyzing immobilized particle arrays.12

Lucifer Yellow CHLucifer yellow CH (LY-CH or LY, Figure 14.3.5) has long been a

favorite tool for studying neuronal morphology because it contains a carbohydrazide (CH) group that allows it to be covalently linked to surrounding biomolecules during aldehyde-based �xation.13,14 Loading of this polar tracer and other similar impermeant dyes is usually ac-complished by microinjection,15 pinocytosis,16 scrape loading,17 ATP-induced permeabilization 18 or osmotic shock 19 (Table 14.1), but can also be accomplished in cell suspensions or with adherent cells by using our In�ux™ pinocytic cell-loading reagent (I14402, see below). Lucifer yellow CH localizes in the plant vacuole when taken up either through what is thought to be anion-transport channe

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CHAPTER 1 CHAPTER 14 Fluorophores and Fluorescent …...Section 14.1 Choosing a Tracer ˜˚.˜ Choosing a Tracer Figure 14.1.1Collage of images of cyanobacteria stained with various