35mmplates:

1. to2mlmediumorPBS,add2ul2mg/mlP.I.6ul5mg/mlFDA
2. R.T.3min
3. Rinse1XPBS
4. leavecellsinPBS.Examinecellsunderthescopeimmediately.

1. IftheP.I.stainingisnotstrongenoughtobepickedupeasilyunderyourscope,use2XP.I.,i.e.,4ul2mg/mlin2mlmedium
2. Afterstaining,needtoexaminethestainingrightaway,otherwise,thegreenstaininggetsdiffused.Youcanleavecellsat4forafewhr.-overnighttoslowdownthediffusion(Ihavetried3T3,donotknowifitworksforneurons)
3. Ref.:K.H.Jones&J.A.Senft(1985)J.Histochemistry&Cytochemistry33:77-79M.Schrammetal.,(1990)PNAS87:1193-1197
4. Thismethodstainsfornon-fixedcells.
5. P.I.:Sigma,dissolveinPBSFDA:Sigma,dissolveinacetone

1. Fixation:
   1. ETOHfixation-givesbrighterP.I.stainingGentlyoverlayovermedia4Xmediavol.ofETOHprecooledto-20R.T.3minGentlymixmedia&ETOHwithpipetR.T.5minor
   2. Paraformaldehydefixation:(8%paraformaldehyde/4%sucrose/inPBS,pH7.2-7.6)Gentlyoverlayovermedia2Xmediavol.of8%paraformaldehyde/4%sucrose/inPBS,pH7.2-7.6gentlytilttheplatestomixR.T.15min
2. Aspirateoffmedia
3. Staining:4ug/ulP.I./0.1%tritonX-100/0.5mg/mlRNaseAinPBSR.T.5minExamineunderthescopeormountwithcoverslipsNote:
   1. P.I.willstainforbothDNAandRNA.ItiscriticaltoincludeRNaseAtoeliminatethecytosolicRNAstainingbackground.IfuseETOHfixation,itislesscriticaltoincludeRNaseAinstainingsoln.
   2. Thiswillstainbothaliveanddeadcells.Alivecellsshouldhaveevenlystainednuclei.Nucleifromapoptoticcellsshowcondensed,orfragmentedmorphology.Cannotdistinguishnecrosis.

1. Fixcells
   1. removemedia,fixw/4%paraformaldehyde/4%sucroseinPBS,neutralpH,RT15-45min
   2. ifcellsarenotadhereingwelltotheplates:Gentlyoverlayovermedia2Xmediavol.of8%paraformaldehyde/4%sucrose/inPBS,pH7.2-7.6gentlytilttheplatestomixR.T.15min
2. wash1XPBS/0.1%tritonX-100,RT5min
3. staincellsw/2.5ug/ulHoeschst33258inPBS/0.1%tritonX-100R.T.5min
4. wash1XPBS/0.1%tritonX-100,RT5min
5. Mountw/coverslips.ExaminecellsuderfluorescencescopeusingDAPIfilter

1. Alivecellsshouldhaveevenlystainednuclei.Nucleifromapoptoticcellsshowcondensed,orfragmentedmorphology.
2. Hoeschst33258,SigmaB-2883(bis-Benzimide),5mg/mlinH2Ostock.Lightsensitive.
3. Hoeschst33258stainspermeablizedcells;Hoeschst33342ispermable,canstainbothfixedandnon-fixedcells.

Morphologically:Alivecells:phasebright

Necrotic:cellswelling,i.e.,enlargedcellbodies,cellmembraneleakage,lysisofcellbody

Apoptotic:roughmembrane,plasmamembraneshrinkage,cellbodyshrinkage,membraneblebbing,nolysisofcellbody

Staining:

1. Trypanblue:deadcellsstainblue.Cannotdistinguishnecroticvsterminallyapoptoticcells
2. FDA/P.I.staining:Alivecellsstainblue,necroticorterminallyapoptoticcellsstainred.Earlyapoptoticcellsshouldnotstainred.
3. P.I.orHoeschststainingoffixedcells:Nucleifromapoptoticcellsshowcondensed,orfragmentedmorphology.
4. Tunnelstaining:commercialkitsavailable.Nucleifromapoptoticcellsshowcondensed,orfragmentedmorphology.
5. DNAladder:NecroticcellsdonotshowDNAladdering;Most,butnotall,oftheapoptoticcellsshowDNAladdering.

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