Materialstobepreparedbeforehand:1)WashingBuffer--0.1%BSAinmedium(DMEMorRPMI)2)BlockingBuffer--0.5%BSAinmedium(DMEMorRPMI)3)Laminin-110-12μg/mlorFN20μg/ml4)96-well-plate5)Crystalviolet(5mg/mlin2%Ethnol)6)1%SDSinH2O7)4%paraformaldehydeProcedures:1)Coat96-well-platewithLaminin-1orFNat37oCfor1hrorat4oCO/N.Leavesomewellsuncoatedasnegativecontrol.2)Washwithwashingbufferfor2times.3)Blockplateswithblockingbufferat37oCinCO2incubatorfor45-60minutes.4)Washwithwashingbuffer.5)Chilltheplatesonice.6)Countcellto4X105/ml.Add50μlcellsineachwell.7)IncubateinCO2incubatorat37oCfor30minutes.8)Shaketheplateat2000rpmfor10-15seconds.Washwithwashingbuffer2-3times.9)Fixwith4%paraformaldehyde.IncubateatRTfor10-15minutes.10)Washwithwashingbuffer.11)StainwithCrystalVioletfor10minutes.12)Washwithwater.13)Turntheplatesupsidedown.Lettheplatesdryupcompletely.14)Add2%SDS.IncubateatRTfor30min.15)Readplateat550μm.
