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Dynamic Flow Assay in a Parallel Plate Flow Chamber
Flowassaysallowvisualizationofcelladhesionunderwell-definedwallshearstress.Thevisualizationofthedifferenteventsofcelladhesioncanbequantifiedbyselectiveimageacquisitionandsubsequentimageprocessing.Flowassaysareuniquelysuitedtotheinvestigationofadhesiveeventswhichoccurveryrapidlyinatimescaleshorterthanthatofmoststaticadhesionassays.Inaddition,eventssubsequenttotheinitialeventscanbestudiedsuchascellstABIlizationandspreADInggivingsomeinsightintothekineticsofparticularcell-cellorcell-substrateadhesivebehavior. Inverted-stagemicroscopeconfiguredforphasecontrastandfluorescenceoperation Objectivelens(6.3x,10x,40x) Stageincubator CellsUSPension(neutrophils,cancercells) Cellmonolayerorcoatedsubstratein35mmdishes Adhesionmedia(culturemediaserum-freewith12mMHepes) Fibronectin(humanplasma) Bovineserumalbumin(BSA) Flowchamberdeckwithgasketandtubefittings 35mmtissueculturedishes Harvardsyringepump Glasssyringestofitonpump(3,5,20,or60cc) Silastictubing Stopcocks Vacuumpump Digitalimageacquisitionandprocessingsystem Displaymonitor Videorecorder CCDcamerawithcontroller Time-dategenerator Computerwithinterfacetoimagesystem Storagedeviceforimages 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.Materials:
Microscopy: BIOLOGical: FlowChamberSystem: Computer/Electronics: Protocol:
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