BD BioCoat™ and BD Falcon™ FluoroBlok™Tips and Techniques

Jeff Partridge, M.S.April 27, 2010

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Overview

Part I BD FluoroBlok™ System Description

Part II Tips and Tricks

Part III Questions and Answers

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BD FluoroBlok membrane

Cross section of an insert system – not to scale

Pore

Basal chamber

Apical chamber

Insert (individual or multiwell)

Base plate

What is the BD Falcon™ FluoroBlokInsert System?

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8 μm pores – visible light

What is the BD Falcon FluoroBlokInsert System?

Light-tight PET membraneDyed membrane blocks light from 490-700 nm while allowing for multiplex detection assays

Choice of membrane pore sizes24 individual cell culture inserts or 24- and 96-multiwell insert plates available in 3 or 8 µm pore sizes

Run homogeneous assays in real time with non-destructive detectionRapid data collection without the need for plate washing or manual cellscraping and countingDetermine migration or invasion of cells in real time

Quantitative detectionUse of fluorescence detection

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What Can You do with the BD FluoroBlokInsert System?

Cell MigrationHaptotaxis – movement in response to a gradient of adhesion sites or ECMsChemotaxis – movement toward a chemical gradientCell motility – chemokinesis, cell movement

Cell Invasion Degradation of a physical barrierPore-occluding BD Matrigel™ Matrix-coated BD FluoroBlok Insert Systems

Co-cultureMultiple cell types in close proximity

A variety of cell types on either BD Falcon or BD BioCoat™ FluoroBlok Insert Systems can be used.

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Insert System Handlingpipettes and plates

A repeating pipettor is recommended for the 24-multiwell or individual insert system.

A multi-channel pipettor is required for the 96-multiwell insert system.

The 96 square-well plate must be kept level to minimize siphoning.

When adding cells to inserts, pipette gently so as not to disrupt the matrix.

Add somewhat slowly, instead of quickly adding directly onto the matrix:this is somewhat easier when using 96-well insert systems.

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Insert System Handlinginsert volumes

Volumes added to apical and basal chambers must match.

Typical chamber volumes

apical basalnominal range nominal range

24-well 250 μL 250-500 μL 750 μL 750-1400 μL96-well 50 μL 30-70 μL 200 μL 200-225 μL

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Insert System Handlingthawing and rehydration of matrix

For BD Matrigel matrix-coated inserts, remove the foil bag from -20°C and allow it to reach RT.

Once thawed, you must use all of that insert system.

For five packs, you may reseal the package and use the unthawed systems.

Buffering system is important.

Rehydration medium must match conditions:medium use CO2 incubatorPBS best to use non-CO2 incubator

*carbonate/bicarbonate-buffered medium would require CO2.

No need to rehydrate uncoated membranes.

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Insert System Handlingcells

Use good aseptic technique.

Use low passage # cells.

Do not use cells that are over-confluent to start.

Use cells that are in log-growth phase for best results.

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Cell Culture Insert Handlingindividual inserts

companion plate (w/notches)

+

=

individual inserts (w/flanges)

insert flanges resting in plate notches

*When using individual cell culture inserts, both the inserts and companion plates must be used.

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Insert System Handlingindividual inserts and multiwells

Be careful when feeding or aspirating individual inserts.

Gently move aside insert; try to keep or replace flanges in notches.

Be extra careful when removing medium from 96-well insert system fill ports.

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Insert System Handlingthe bubble effect

Bubbles should be eliminated at all steps.

Chemoattractant should be added to the bottom chamber via the access port.

To minimize bubbles, add to the apical chamber then to the basal chamber.

bubble under insert – will influence reading cells may not migrate or stain = bad

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What Format Should I Use?platform choice and scalability

Wide choice of formats:Cell Culture Inserts (clear)Multiwell Insert Systems (clear)BD FluoroBlok cell culture insertsBD FluoroBlok multiwell inserts

24-Multiwell Insert System96-Multiwell Insert System

Tumor Invasion System (BD Matrigel matrix-coated)24 Individual Cell Culture Inserts (clear) – BD Matrigel matrix invasion chamber24 Multiwell Tumor Invasion System96-Multiwell Tumor Invasion System

Angiogenesis System: Endothelial Cell Migration (human fibronectin coated)24 Individual Cell Culture Inserts (clear)24 BD FluoroBlok cell culture inserts24 Multiwell Insert System96-Multiwell Insert System

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What Pore Size Should I Use?platform choice and scalability

Application Pore sizeTransport and Permeability 0.4 µm, 1 µm

Drug transport across epithelial cell monolayer (i.e. Caco-2)

Co-culture and cell monolayer 0.4 µm, 1 µm, 3 µm, 8 µmCell-cell paracrine interactionsHuman endothelial monolayer permeabilityMarine phytoplankters

In vitro model systems 0.4 µm, 1 µm, 3 µmHuman epidermal modelHuman urothelium modelBlood brain barrier

Cell Migration 3 µm, 8 µmLeukocytes Endothelial cells FibroblastsNeutrophils Tumor Cells OsteoblastsSmooth muscle cells PBMCs

Cell Invasion 3 µm, 8 µmTumor Cells Fibroblasts Epithelial cells

Note: This is not an exhaustive list of all cell types and applications.The appropriate pore size is cell type and application specific.

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Insert System Useassay setup

Ensure that you know where your controls are and what they should be:chemoattractant {serum, VEGF, etc.}no chemoattractantno cellsdrug vehiclestain only

No one setup is better than anotheras long as you know where your samples and controls are.

controlcontrolcontrolcontrolcontrolcontrol

testtest

controltest 4

controltest 3

controltest 2

controltest 1

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Insert System Usecalculation of invasion %

Calculation examplesmigration represents 100% of cells coming thru the membrane

can compare invasion as a fraction of that 100%

Why?not all cells will necessarily pass through the insert.

Other examplescan also use increase over a control.

Image Reference – Partridge, J., Flaherty, P. (2009). An In vitro FluoroBlok Tumor Invasion Assay. JoVE 29. http://www.jove.com/index/details.stp?id=1475, doi: 10.3791/1475

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Insert System Usescreening example

How many replicates do you need?

We can use Z’ to assist.

Two replicates should be suitable for screening in this system.

Tumor Invasion System - Z' values96 multiwell

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replicate number

Z'

average Z' = 0.66

Tumor Invasion System - Z' values24 multiwell

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replicate number

Z'

average Z' = 0.70

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BD FluoroBlok InsertsDo You Need an ECM Coating?

www.bdbiosciences.com

Extracellular matrix (ECM) coating may be needed by your cells.

Some cells require ECMs for attachment or signaling.

Choice of ECM and coating conditions needs to be optimized:time, temperature?for immediate use or to be stored and used later?

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BD FluoroBlok Insertsself-coating tips

Consider choice of buffers.

Consider coating volume and thickness.

Swirl coating solution in well.

Be sure when coating you remove all bubbles.

Reconsider fresh coat then use vs. make and store for later use.

Consider the use of BD BioCoat Inserts.

The underside of membranes can be coated.

BD FluoroBlok membrane, 8 µm pore size

Pore occluding BD Matrigel Matrix layer

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Osteoclast Migration Toward ECM Substratesself-coating example

Spessotto P., et al., Journal of Cell Biology, 158(6):1133 (2002).

FLG 29.1 cell line, human preOC, induced to differentiate by TPA

Underside of 8 µm inserts coated with ECM substrates at 20 µg/mL

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BD FluoroBlok Insert Systemcell labeling dyes

Any fluorescent dye derived from the fluorescein, rhodamine and cyanine families can be used with this system. The emission wavelength must be between 490 – 700 nm.

Spectrum image from http://en.wikipedia.org/wiki/Image:Srgbspectrum.png under GNU free documentation license.

— your dye here —

Ultraviolet-inducible dyes tend to be incompatible with the BD Falcon FluoroBlok insert since they tend to emit light in the blue range.

Can I multiplex?Yes! with proper separation between the dyes.

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BD FluoroBlok Insert Systemdyes

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BD FluoroBlok Insert Systemdyes

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interneurons granule cells interneurons granule cells

Cells plated on top of insert (A,E)

Migrating neurons (B,C,F,G)MAP2 stained somata and dendrites (red)DAPI stained nuclei (blue)

1 μm pores visible with some DAPI stained nuclei (blue) that have migrated thru pores

BD FluoroBlok Insert Systemmultiplexing

Hassoun, A., et al., J Neurosci. Methods. 166(2):178–194 (2007).

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BD FluoroBlok Insertsdetection instruments

You will need:A fluorescent plate reader with bottom-reading capability

- AND -An inverted fluorescent microscope for confirmation and troubleshooting

- OR -

A fluorescent imager, i.e., BD Pathway™ Bioimager or Cellomics HCS ArrayScan

BD Pathway 855 High-Content Bioimager

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BD FluoroBlok Insertsdetection instruments

Plate readers compatible with BD FluoroBlok insert systems include:BioTek

Synergy (4, 2, HT), FLx800

BMG LABTECHPHERAStar (Plus), OPTIMA, POLARStar/FLUOStar (Omega, Galaxy)

MDS Analytical TechnologiesSpectraMax readers (Multimode M5/M5e, M2e; Gemini EM)

PerkinElmerEnVision, Victor series, HTS 7000+

TecanSafire2, GENios, SpectraFluor Plus

Thermo Scientific Cellomics HCS ArrayScanFluoroskan Ascent

…and many more!

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BD FluoroBlok Insertsdetection instruments

Plate readers not 100% compatible with BD FluoroBlok insert systems include:MDS FlexStation II

can only be used if the carrier device is removed 24-Multiwell format only

MDS Analyst HT and Analyst GTare compatible with the 96-Multiwell formatnot the 24-Multiwell format, as the door is too short

PerkinElmer Fusion beam size is too large for 24-well platesautofluorescence

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BD FluoroBlok Insertsplate reader set-up

Confirm the plate map supplied with the reader or input the correct coordinates and well diameter into the plate map.

Make sure the correct diameter is used: 6.5 mm for 24-well and 3.2 mm for 96-well.

Plate reader set-up guidewww.bdbiosciences.com/external_files/dl/doc/tech_bulletin/live/web_enabled/tb436.pdf

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BD FluoroBlok Insertsplate reader set-up

Y-line X-line 6×6 square 7×7 ring 7×7 circle

Single or multiple reads per well?for 24-multiwell, multiple reads are generally availablesingle read focuses on center of the membranemultiple reads use reader-specific pattern (within-well average)overall CV improves with multiple reads, however it will take

longer to read a plate

Read areas from Tecan SAFIRE2 (24-multiwell)

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BD FluoroBlok Insertsplate reader tip

Ensure each plate is properly placed into the plate carriage at the A1 position.

A1 plate carriage

FBM insert

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Cell Labeling MethodsWhat should you use?

Pre-Labelinglabeling cells in vitro prior to assay

Post-labelinglabeling cells on the underside of membrane following migration or invasion

Intrinsically-labeled cellsexpressing Green Fluorescent Protein or analogs (e.g., transfected)

Underside of membrane showing SYTO24 (nuclear stain) labeled HT-1080 cells (green) which have migrated.Pores (yellow) are also visible.

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BD FluoroBlok Insertshow to pre-label cells

Generic pre-label protocol

Stain cells when they are attached to flask

Add dye at appropriate concentration for 30 – 60 min

Rinse monolayer with DPBS

Remove cells as usual (trypsin)

Wash 2X then perform assay

DiIC12(3) labeled HT-1080 cells (red) which have invaded (left) or migrated (right) through Tumor Invasion System.

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BD FluoroBlok Insertskinetic assays

Q: How do I run a kinetic assay?

A: You need to pre-label and either take multiple manual readings or use a plate reader that has both temperature and atmospheric capabilities.

Two approaches:

PC3-GFP cells were pre-labeled with SYTO 82,plated in BD FluoroBlok invasion chambers (self-coated BD Matrigel matrix)

Real-time analysis of tumor cell invasion with aFLUOstar OPTIMA plate reader enclosed in anAtmosBag (5% CO2), reader set at 37°C *

HT-1080 and 3T3 pre-labeled with 5 µg/mL DiIC12(3) and plated in a BD BioCoat tumor invasion system

Assay was run for 24 hours in a standard incubatorwith readings taken at various time points †

†Partridge, J. et al., Society of Biomolecular Screening 2005 (conf.).Quantitative and High-throughput Screening of Tumor Cell Invasion Using a Cell-based model.

*BMG Labtech Application Note 144 (12/2006)

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BD FluoroBlok Insertskinetic assays

Fig. 3. Using AtmosBag, automated read*

Fig. 4. Standard incubator, read manually*

kinetic assay

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tim e (h)

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3T3

Standard incubator, read manually †

†Partridge, J. et al., Society of Biomolecular Screening 2005 (conf.).Quantitative and High-throughput Screening of Tumor Cell Invasion Using a Cell-based model.

*BMG Labtech Application Note 144 (12/2006)

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BD FluoroBlok Insertskinetic assays and optimization

THP-1

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lTime course of MCP-1 induced chemotaxis in THP-1 and monocytesPre-labeled cells in the inserts were incubated with 25 nM MCP-1 in the bottom chamberBottom fluorescence was measured at varying time pointsData are means ±SD from a typical experiment (n=4 wells)

BD Biosciences Technical Bulletin #457

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BD FluoroBlok Insertstips – drug studies

DMSO can be used without adverse effect at concentrations up to 2%.

Some drug compounds adhere to plastics:you could use BD Gentest™ enhanced recovery plates for 24-multiwell and 96-multiwell inserts.

Understand the compounds you are using:solubility – will it remain in solution? at what pH?

Do you need to add drug to both apical and basal chambers?

DMSO has no significant effect on Tumor Cell Invasion

0200400600800

10001200140016001800

2% 1% 0.50% 0.25% 0.13% 0%

[DMSO]

RFU

HT10803T3

doxycycline IC50 (μm)invasion migration

Post-label 69 45

Pre-label 64 56

RCFP 72 48

labeling method did not effect IC50

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Mastyugin, V., et al., J Biomol Screen. 9(8):712–718 (2004).

HUVEC in 24-multiwell Angiogenesis System

BD FluoroBlok Insertshigh-content screening assay

better signal-to-background ratio using nuclear stain / HCS than calcein AM labeling / plate reader

EC50 24-well 96-well

AAL993 25 nM 25 nM

data in line with published results

24-multiwell insert system 96-multiwell insert system

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BD FluoroBlok Insertsco-culture

eGFP-10T1/2 cells added to apical side; HUVEC seeded on base plate

NOS inhibitor (L-NMMA) reduced migration of mural cell precursors toward HUVEC

Satoshi Kashiwagi, et al., J. Clin. Invest. 115:1816–1827 (2005).

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Pre-labeled for 15 minutes in flask

Cells migrate through and then fall from insert and collect beneath insert

BD FluoroBlok Insertsuse of non-adherent cells

neutrophil chemotaxis

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Some Selected Q&Awhy isn’t my assay working?

Q: Why isn’t my assay working?A: Low invasive cells

donor effect (see next slide)

Q: What can I do?A: Try pre-label to get kinetic data

increase chemoattractant concentration (5% 10%)increase invasion time (24h 72h or more)increase cell density (should try curve initially)

typical seeding density24-well: 25,000 - 50,000 cells/well96-well: 10,000 - 20,000 cells/well

you may need to optimize stain concentration

you might need to use a different chemoattractant

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BD FluoroBlok Insertsdonor variability

Human MSCs pre-labeled with CellTracker Greenworking [fMLP] established

Anand Viswanathan, et al.,Stem Cells 25:1263–1269 (2007).

Human MSCs from different donors pre-labeled with CellTracker Green100 nM fMLP as chemoattractant

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BD FluoroBlok Insertsassay optimization

Hassoun, A., et al., J Neurosci. Methods. 166(2):178–194 (2007).

Neuron migration thru BD Matrigel matrix in response to motogen (BDNF)

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BD FluoroBlok Insertsassay optimization tips

Effect of MMP inhibitor 1'10' Phenanthroline on HMVEC Invasion

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lVEGF(4n

g/ml)

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VEGF(4ng/ml)+ 1'10' Phenathroline

Fluo

resc

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nits

BD Matrigel matrix-coated BD FluoroBlok 3 µm pore size, 24 multiwell insert

Optimized for endothelial cell invasion

HUVEC MigrationHFN coated vs. uncoated

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Human fibronectin-coated BD FluoroBlok 3 µm pore size, 24- and 96-multiwell inserts

Optimized for endothelial cell migration

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BD FluoroBlok Insertshow to test the system

Acquire 3 or 8 µm BD FluoroBlok™ insert system in the format of your choice.

Label HUVEC (3 µm) or HT-1080 (8 µm) in a TC flask with 5 µg/ml of calcein AM / HBSS [1 h, 37°C, 5% CO2].

Wash the cells and seed in basal medium.

Add 5% FBS in basal medium as the chemoattractant.

Incubate for 4 hours at 37°C, 5% CO2.

Read the insert system on your plate reader.

Confirm by observing the cells on the membrane using an inverted fluorescent microscope.

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Selected Q&Aremoval of cells

Q: Can cells be removed from the underside of membrane if attached?

A: Yes, use trypsin or cell scraping.

Try your standard protocol basedon cell type and conditions.

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Selected Q&Acondensation

Q: Will condensation on the lid affect my experiment or the system?

A: No. This is a bottom-reading system.

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Additional Thanks to . . .

Bill Fiore

Paula Flaherty

Kevin Kelly

Amyntrah Maxwell

Shabana Islam

Amy Laws

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Upcoming Webinar

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May 12, 2010

In Vitro Models for Studying Angiogenesis

www.bdbiosciences.com/webinars

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Questions?

Technical Support:

In the U.S.tel: 877.232.8995e-mail: labware@bd.comOutside the U.S.e-mail: help.biosciences@bd.comVisit bdbiosciences.com/webinarsFor research use only. Not intended for use in diagnostic or therapeutic procedures. BD, BD Logo, and all other trademarks are the property of Becton, Dickinson and Company. ©2010 BD

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Selected References

general tumor invasion protocolPartridge, J., Flaherty, P. (2009). An In vitro FluoroBlok Tumor Invasion Assay. JoVE 29. http://www.jove.com/index/details.stp?id=1475, doi: 10.3791/1475

kinetic invasion assay, multiplexing*BMG Labtech Application Note 144 (12/2006)

invasion/migration drug screening (doxycycline)BD Biosciences Technical Bulletins #441 and 442.

HCS screening of migration assayMastyugin, V., et al., J Biomol Screen. 9(8):712–718 (2004).

neuronal motogen screeningHassoun, A., et al., J Neurosci. Methods. 166(2):178–194 (2007).

Time Course of MCP-1 Induced Chemotaxis in THP-1 and Primary MonocytesBD Biosciences Technical Bulletin #457

DiI Labeled Osteoclast Migration Toward ECM SubstratesSpessotto P., et al., Journal of Cell Biology, 158(6):1133 (2002).

NO-Mediated MigrationSatoshi Kashiwagi, et al., J. Clin. Invest. 115:1816–1827 (2005).

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Selected References

Chemotaxis of Undifferentiated and Transfected HL-60 cells using 3 µm BD FluoroBlok InsertsChristophe, et al., J. Biol. Chem. 276:21585 (2001).

Human MSC ChemotaxisAnand Viswanathan, et al.,Stem Cells 25:1263–1269 (2007).

Effects of Eph B4 Receptor Activation Primary Human Microvascular Endothelial CellsJena J. Steinle, et al., J. Biol. Chem. 277(46):43830 (2002).

A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening AssaysZhang, et al., J Biomol. Screen 4:67 (1999).

CD155 (Polio Virus Receptor) plays a key role in cell motility during tumor cell invasion and migrationSloan, et al., BMC Cancer, 4:73 (2004).

Chemotaxis Study: To Determine Therapeutic Potential of SDF-1 in Hematopoietic Stem Cell MobilizationPelus, et al., Letters in Drug Design & Discovery, 1:126 (2004).