Manydifferentmoleculeshavebeendescribedtopromotecelladhesionincludingseveralcellsurfacecarbohydrate-bindingproteins.Measuringcelladhesionintheconvenient96-wellmicrotiterplateformatisdifficultduetotheshearforcesgeneratedbywashingthewells.Thisprotocolintroducestheuseofaliquid-filledwashchamberthatseparatesunboundcellsbygravitytherebyeliminatinguncontrolledshearforcesandpassageofadherentcellsthroughaliquid/airinterface.Thecellsareloadedwithafluorescentdye(6-carboxyfluoresceindiacetate)fordetectionalthoughothermethodssuchasrADIoactivelabelsmaybeused.Thisprotocolisalsousefulforassayingmoleculesthatpromoteorinhibitcelladhesion. TestcellsinsUSPension Testcelladhesionmolecule 6-carboxyfluoresceindiacetate Microtiterplate(96-well) Automatic96-wellplatewasher(optional) Microtiterplatefluorescenceplatereader HEPESCaMgbuffer:(0.05MHEPES,0.15MNaCI,1mMCaCl2,1mMMgCl2,pH7.4) Bovineserumalbumin(BSA) RPMI1640culturemedia Fluorescencemeasurementsystemformicrotiterplates 1.Coat96-wellmicrotiterplatewiththecelladhesionmoleculebyincubationofeachwellwith200ngofcelladhesionmoleculesin100祃ofHEPESCaMgbufferfor2hrs.at37癈. 2.Blockfurthernon-specificbindingtotheplasticwellsbytheadditionof100祃of2%BSAinHEPESCaMg/well.Incubateatroomtemperaturefor1hr. 3.WashthemicrotiterplatewithHEPESCaMgbufferbyhandorbyusinganautomatic96-wellplatewasher. 4.Fillwellswith100祃ofHEPESCaMgbuffercontaining1%BSAoraddtestinhibitorsofcelladhesionin100祃ofHEPESCaMgbuffercontaining1%BSAtoeachwellandincubatefor2hrs.atroomtemperature. 5.Duringincubationloadtestcellswith6-carboxyfluoresceindiacetate(6-CFDA)asfollows: A.WashcellsuspensionwithRPMI1640 B.Resuspendcellsin4.8mlofRPMI1640 C.Add200祃ofa1mg/mlsolutionof6-CFDAdis-solvedinRPMI1640 D.Incubateat37癈for30mins. E.Washcells3timeswithHEPESCaMgbuffer F.Adjustthecellconcentrationto2x106cells/ml. 6.Add100祃of6-CFDAloadedcells(2x106cells/ml)tocoatedwellscontaining100祃ofHEPESCaMg. 7.Incubateatroomtemperaturefor20min. 8.PlacemicrotiterplateinstaticcelladhesionWashChamberfilledwithHEPESCaMgbufferandclosechambertoeliminateanyairinterfaceandleakage. 9.Invertthechamberandallowunboundcellstofalloutofwellsfor6min.atroomtemperature. 10.Standthewashchamberonend,removethegasketedtop,andwithforcepsgentlyremovetheplateata15degreeangletoensureliquidremainsinwells. 11.MeasurefluorescenceusingamicrotiterplatereadersuchastheCytofluor2350fromPerseptiveBiosystems,Inc.Materials:
StaticcelladhesionWashChamber Protocol:
