DescriptionCellStainingforSenescence-Associatedâ-Galactosidase(SA-â-Gal)ActivityProcedure1.Carefullyremovethegrowthmediafromthecellculture.2.AddanequalvolumeofPBSandgentlymixthePBSsolutionaroundtowashoutthecellculturemedia.3.RepeatthePBSwashofthecellstwomoretimes.4.DiscardthePBSwashsolution.5.AddFixativeSolutiontothecellsandincubateatroomtemperaturefor3to5min(seeHint#2).6.WashcellsasbeforeinPBStwotimes.7.Addbetween1to2mlofStainingSolutionper35mmculturedishorcoverslide.8.Incubateforatleast2hrat37°C(donotincubateinaCO2incubator;seeHint#3).RecipesStainingSolution1mlof100mMK3Fe(CN)6(finalconcentration5mM)40ìlof1MMgCl2(finalconcentration2mM)*Addjustbeforeuse600ìlof5MNaCl(finalconcentration150mM)PrepareStainingSolutionthedayofexperiment4mlofCitricAcid/PhosphateBuffer(finalconcentration30mM)1mlof100mMK4Fe(CN)6(finalconcentration5mM)1mlofX-GalSolution*(finalconcentration1mg/ml)CellFixativeinPBS2%(v/v)Formaldehyde0.2%(w/v)Glutaraldehyde1MMgCl25MNaClX-GalSolutioninDMF(CAUTION!seeHint#1)20mg/mlX-Gal(5-bromo-4-chloro-3-indolyÂ-D-galactopyranoside)Store-20°CX-GalisnotstableinwaterandshouldbeaddedtotheStainingSolutionjustbeforeuse100mMPotassiumFerricyanide100mMPotassiumFerricyanide(K3Fe(CN)6)CAUTIONseeHint#1100mMPotassiumFerrocyanide100mMPotassiumFerrocyanide(K4Fe(CN)6)CAUTIONseeHint#1CitricAcid/PhosphateBuffer0.1MCitricAcid0.2MSodiumPhosphateDibasic(Na2HPO4)ProperpHisVERYimportantpH6.0PBSpH7.22.7mMKCl4.3mMSodiumPhosphateDibasic(Na2HPO4)1.8mMPotassiumPhosphateMonobasic(KH2PO4)137mMNaClSuppliesTips1.CAUTION!Thissubstanceisabiohazard.Consultthisagent"sMSDSforproperhandlinginstructions.2.Cellscanalsobefixedin3%(v/v)FormaldehydeinPBS;howevertheFixativeSolutionpreservesmorphologybetter.3.Abluecolorisvisiblewithin2hr;however,stainingismaximumatbetween12to16hr.Bluestainisindicativeofsenescence-associatedâ-galactosidaseactivity.