CELLADHESION

1)Dissolve/dilutecoatingsubstrateinddH2Oat4C.Acommonworkingdilutionforthelaminin-1positivecontrolandBSA(SigmaA8412)negativecontrolis40ug/ml.ForSN-peptide,use20uM(MWofSN-peptideis2412)forplateauor2.5-5uMforhalf-maximaladhesion.ForfragmentE8orlaminin-1plateauandhalf-maximaladhesionareusuallyachievedat0.5and0.05uM,respectively.

2)Addcoatingsolution(100ul/well)withmultiPipettertowellsofa96welltissuecultureplate(Costar#3595),cover,andplaceat4Covernight.Coatintriplicateorquadruplicate.

3)Invertplateandshakeoutcoatingsolution.Pulloffremainingcoatingsolutionfromeachwellwithayellowtippipetter.

4)Dilute7.5%BSA(SigmaA8412)to1%inddH2O.Add100ul/wellwithmultipipetter,coverandplaceat4Cfor4hrs.Thaw10xtrypsin/EDTA(thendiluteto1xinPBS),warmPBSandserum-freemedium.

5)Inlast45minofblock,pulloffmediumfromcellsinT75flask,andaddserum-freemedium.Replaceinincubatorfor30min.Subsequently,pulloffmedium,washwithPBS,add1xtrypsin/EDTAfor1-3min,pulloffreleasedcells,washflaskwith20mlofserum-freemedium,pelletcellsin40mlofserum-freemedium,makeupin6mlofserum-freemediumandcount(15ulofsUSPendedcellsplus15uloftrypanblue;add15ultoeachsideofhemocytometer;cell#/ml=combinedcountfrombothsidesx104).Dilutecellsto2.0x105/mlinserum-freemedium.

6)InvertplateandshakeoutBSAblockingsolution.Pulloffremainingblockingsolutionfromeachwellwithayellowtippipetter.

7)PourcellsinReagentReservoir(Costar#4870),rocktosuspend,remove100ul/wellwithmultipipetterandaddtowells.Repeatrock/resuspensionpriortoremovingcellsuspensionforeachrow.Placeinincubatorfor30-60min(37C).8)Examineplateininvertmicroscope.Photographselectedwellsifdesired.Invertplategentlyontoanabsorbentdiaperpad.Pulloffremainingcellsolutionfromeachwellwithpipetter.

9)Withmultipipetter,slowlyadd100ul/wellofserum-freemediumdownthesideofeachwell(tiltplate;PBSisnotrecommendedforthiswash).Invertplategentlyontoanabsorbentdiaperpad.Pulloffremainingwashsolutionfromeachwellwithpipetter.

10)Slowlyadd100ul/wellofserum-freemediumdownthesideofeachwell.Examineplateininvertmicroscope.CellsinBSAnegativecontrolwellsshouldberare(ifnot,repeatwash).AdherentcellsinSN-peptidewellsremainmainlyroundedorslightlyspread.AnexceptionisM2melanoma,whichspreadsrapidlyonSN-peptide.Adherentcellsinlaminin-1wellsshouldbeallspread.Invertplategentlyontoanabsorbentdiaperpad.Pulloffremainingwashsolutionfromeachwellwithpipetter.

11)Withmultipipetter,slowlyadd100ul/welloffreshlydiluted1%glutaraldehydeinPBS.Fixfor10minatroomtemp.Invertplategentlyontoanabsorbentdiaperpad.Pulloffremainingfixsolutionfromeachwellwithpipetter.

12)Withmultipipetter,add100ul/welloffreshlyfiltered(use0.2umsyringefilter)crystalviolet(0.1%inddH2O;Serva#27335).Stainfor25minatroomtemp.Invertplateontoanabsorbentdiaperpad,thenwashplategentlybyimmersioninaplastictraycontainingtapwater.Invertplateontoanabsorbentdiaperpad.Pulloffremainingwashfromeachwellwithpipetter.Reimmerseinfreshtapwater.Invertplateontoanabsorbentdiaperpad.Pulloffremainingwashfromeachwellwithpipetter.Repeatanadditionaltimeifrequired.Allowtodryfor5-10minatroomtemp.

13)Withmultipipetter,add50ul/wellof0.5%TritonX-100(dilutedinddH2O).Allowtosolubilizeovernightatroomtemp.inadrawer.ReadatOD595.BSAbackgroundshouldbelessthan0.1OD.Lamininvalueshouldbeabout1.0OD.PlateauSN-peptidevalueisusually70-80%oflaminin.