Flowassaysallowvisualizationofcelladhesionunderwell-definedwallshearstress.Thevisualizationofthedifferenteventsofcelladhesioncanbequantifiedbyselectiveimageacquisitionandsubsequentimageprocessing.Flowassaysareuniquelysuitedtotheinvestigationofadhesiveeventswhichoccurveryrapidlyinatimescaleshorterthanthatofmoststaticadhesionassays.Inaddition,eventssubsequenttotheinitialeventscanbestudiedsuchascellstABIlizationandspreADInggivingsomeinsightintothekineticsofparticularcell-cellorcell-substrateadhesivebehavior.

Materials:

Protocol:

CellMonolayerPreparation

1.Coatdisheswithhumanfibronectin(FN)byadding1mlof5.5礸/mlFNsolutiontoeachdish.

2.Incubatefor30min.atroomtemperature.

3.Aspiratesolutionoutofeachdish.

4.Add2mlofcellsuspension(Huvecs,CHOcells)toeachdishatseedingdensityof0.5-3.0x10⁵cells/mldependingontimerequiredtoreachconfluence.

5.After1-5days,usedisheswithconfluentmonolayerinflowassay.Feedcellsevery2-3days,butusuallynotwith48hrs.offlowassay.

CoatedSubstratePreparation

1.Outlineacoatingregion(5mmdiameter)forcoatingsubstrateinthecenterofeachdishwithmarkingpen.

2.Add20祃ofsolutioncontainingsubstrateataconcentrationof10礸/mltocoatingregion.Incubate1hr.

3.Aspirateoffliquid,add20祃of1%BSAtoblockfor1hr.

4.AspirateoffBSA,add20祃ofinhibitorfor1hr.

5.Dishesarereadyforuseinflowassay.

FlowAssayUsingParallelPlateFlowChamber

1.Turnonstageincubatorandwarmadhesionmediato37癈1hr.priortobeginningflowassay.

2.Assembleflowsystemapparatusconnectinginlet,outlet,andvacuumlinestotheflowchamberdeck.Fillsystemwithmediaandremoveallairfromsystem.

3.Fillinletreservoirwithcellsuspension.Forassaysusingcellmonolayers,10⁶cells/mlisrecommended.Forcoatedsubstrateassays,use10⁵cells/ml.

4.Attachdishtoflowchamberdeckbyholdingthedeckinverted,placeasmallbubbleofmediaonflowpatharea,thenplace35mmdishonthedeck.Vacuumwillholddishondeck.Makesuredishwasattachedwithnoairbubblesintheflowpath.

5.Placeassembledchamberonmicroscopestage.

6.Initiateflowofcellssyringepumpconnectedtooutletflowchamberatashearstressintherangeof0.1-4.0dynes/cm².

7.Allowcellstoflowforsufficienttimetogetanadequatenumberofcellsinteractingwiththecellmonolayerorcoatedsurface.Generally3-10min.isused.

8.Beginimageacquisition.Collectimagesat7-10locationsonthedish.Generally3dishesatagivenexperimentalconditiongivesenoughdatatoshowstatisticaldifferencesbetweentreatments.

9.Afterimagesareacquiredonalldishes,performimageanalysistoquantifytheflowassay.

References:

(1)Lawrence,M.B.,McIntire,L.V.,Eskin,S.K.,(1987),Effectofflowonpolymorphonuclearleukocyte/endothelialcelladhesion,Blood70:1284-1290.

(2)Patton,J.T.,Menter,D.G.,Benson,D.M.,Nicolson,G.L.,McIntire,L.V.,(1993),Computerizedanalysisoftumorcellsflowinginaparallelplatechambertodeterminetheiradhesionstabilizationlagtime,CellMotilityandtheCytoskeleton26:88-98.

(3)Jones,D.A.,Abbassi,O.,McIntire,L.V.,McEver,R.P.,Smith,C.W.,(1993),P-selectinmediatesneutrophilrollingonhistamine-stimulatedendothelialcells,BiophysicalJournal65:1560-1569.

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