thawcellsfromliquidnitrogen,growin75ccflask(T75)inFischer"smediumMM(maintenancemedium)untilcontentgetscloudyandstartstoturnyellow(usuallywant~6x106cellsperT75),assaywillbeperformedin24-well-plateatconcentrationof100,000cells/well,eachsampleintriplicate. centrifugecells,bringbacktovolumewithFischer"sSM(stationarymedium).ItisimportanttoremoveallofMMfromcellssothatnegativecontrolislow.Ifwanted,cellscanberinsedinSMandspunagain. spincells,bringtosmallervolumewithSMandcount,willwant100,000cells/0.5mL/well;allproteinsandcontrolsdoneintriplicate.Towellscontaining0.5mLcellsUSPensionadd: incubateat37癈for72hrs,thenreadoncellcounter(SysmexMicrocellcounterCC110) add400礚oftestsustanceto10mLHematall,countoncounter.ReADIngofcellcountx57,000iscellnumberin1mLoriginalcellculturebeforestart:
Onedaybeforeassay:
Dayofassay:
Cellcounter:
