Reagents Cellstobestudiedexpressinggreenfluorescentprotein(GFP).NotethatthesamecelltypewithoutGFPisneededasacontrol. 1XPBS 2%Bufferedformaldehydesolution(seerecipe) 70%Ethanol Propidiumiodidestocksolution(1mg/mlinPBS) DNAse-freeribonucleaseA 12X75mmculturetubes Vortexmixer Waterbathat37oC Method Fixcellswithformaldehyde 1.Countcells. 2.Placeapproximately106cellsintoa12x15mmtesttubeandwashthemoncewithPBSbycentrifugationfor5minat300xgat2-8oC. 3.Removesupernatantbyaspirationorrapiddecantingandadd500mlofcoldPBStothecellpellet.Mixgently.Add500mlofcold,buffered2%formaldehydesolutionandmixagain.Incubateat2-8oCfor1h. PermeABIlizecellswithethanol 4.Spincellsdownbycentrifugationfor5minat300xgat2-8oC,removesupernatantbyaspirationorrapiddecanting,washoncewithcold1XPBS,thenadd1mlof70%ethanolat–20oCdrop-wisetothecellpelletwiththetubesittingonavortex.IncubatecellsUSPensionovernightat2-8oC. 5.Spincellsdownbycentrifugationfor5minat300xgat2-8oC,removesupernatantbyaspirationorrapiddecantingandadd1mlofasolutioncontaining40µg/mlofPIand100µg/mlofribonucleaseA.Incubatecellsuspensionat37oCinthedarkfor30min.Ifneeded,filtersamplesthroughanylonmeshtoremoveclumpsbeforeacquisitionontheflowcytometer. PreparationofBuffered2%FormaldehydeSolution Add2gparaformaldehyde(polysciences,Inc.)to100mlPBS.Heatthesolutionto70oCinafumehooduntiltheparaformaldehydegoesintosolution(approximately1h).Cooltoroomtemperature,checkpHandadjustto7.2with0.1MNaOHor0.1MHCl.Storeat2-8oCprotectedfromlight.Thesolutionisstableforatleast1month.CheckpHperiodically.Donotheatthesolutionabove70oC.Forbestresults,useonlyverypurepreparationsofparaformaldehyde(i.e.,electronmicroscopygradefromPolysciences).
