DescriptionAllowsonetotestthecapABIlityofadherentcellstosurviveandreplicatefollowinginsultwithchemicalsorrADIation.Procedure1.Growthecellsinregularmediato70%confluenceandtreatthecellswiththeappropriatechemicalorradiationdose.Besuretoincludeanontreatedcontrol.2.Attherequiredendpoint,trypsiniseandcountthecellsasusual.3.Re-seed1000cellsintoanew60mmor100mmtissueculturedish(induplicate)andincubatefor9days.Freshmediashouldbeaddedatday5.(SeeTip#1)4.Atday9(orwhenthecellcolonieshavegrowntoapprox50cells)removethemediaandadd1ml(for60mmdish)or2ml(for100mmdish)ofClonogenicReagent.Leaveatroomtemperaturefor45minutes.5.WashthecellstwicewithPBSandcountthebluecolonies.6.Thedatacanthenbeexpressedaspercentsurvivalrelativetothecontrol:((averagetreatedcount)/(averagectrlcount))x100RecipesCLONOGENICREAGENT50%Ethanol0.25%1,9-dimethyl-methyleneblueSupplies1,9-dimethyl-methylenebluefromSigmaAldrich(Cat.#34,108-8)TipsTip#1:Seededatsuchalowdensitysomecellshavedifficultyadheringtotheplates.ItmayhelptouseCellPlusplatesfromSarstedttoincreaseadherenceORpolylysinetreattheplates.
