FIXATION and DNA Staining for Cell Cycle Analysis
来自 : 蚂蚁淘
Background
ThismethodofDNAstainingutilizesethanoltofixthecellsandpermeABIlizethemembrane,whichallowsthedye(PropidiumIodide)toenterthecells.PropidiumIodide(PI)isaDNA-bindingFluorochromethatintercalatesinthedouble-helix.Ribonuclease-Aisusedtoeliminatethestainingofdouble-strandedRNA.Generally,themethodcanbecombinedwiththeproceduresfordirectlabelingorindirectlabelingofcellsurfaceantigens.UseonlyFITC-labeledantibodiessincePIemitsintheorangetoredregionofthespectrum(FITCemitsgreen).Anddonotfixthecellswithparaformaldehyde-startwiththisprotocolafterthelastwashstepinthedirectorindirectimmunofluorescenceprotocols.
Thefluorescencefrommanyantibody-labeledcellsurfaceantigensisoftenpartiallydiminishedfollowingthisprocedure.Otherantigensmaybetotallydenaturedandallfluorescencemaybelost.Itisbesttotestforthisbeforebeginningalargedual-staining(cellsurfaceandDNA)experiment.
Materials
- DNAStain:5milligramsofPIin100millilitersof1.12%(w/v)ofsodiumcitrate.
- RNAsesolution:500unitsperml.in1.12%(w/v)ofsodiumcitrate.
UseahighlypurifiedRNAsetoavoiddestructionfothecellsbycontaminatingproteasesand/orDNAses.Otherwise,theRNasesolutioncanbepurifiedbyheatingto75degreescelciusfor30minutes.Cooltoroomtemperaturebeforetreatingthecells.
- 100%ethanol.
- Fetalornewbornbovineserum,(optional)forremovalofdebris.
- CellsinsUSPension,countedandviability-checked.Theprocedureiswrittenforaliquotsof1millioncells,butyoucanscaleitupasrequired.ThecellsshouldbesuspendedinculturemediaorPBSwithoutserum,astheethanolfixationstepwillpercipitatetheproteinsintheserum.
Thisproceduredoesnotnecessarilyrequire100%viablecells,butgenerallythemoredeadcellsyouhave,themoredebrisandmulti-cellaggregatesyoucanexpect.
Equipment
- Centrifuge.YoushouldknowhowtheRPMtranslatesintoG-force.
- Precisionadjustablemicropipet.Itshouldcovertherangefrom100to1000microliters.
- Pasteur-typetransferPipettes,eitherglassorplastic.
- Cottonswabs.
- Vortexmixer.
- 37degreeC.waterbath.
- 12x75mmpolystyrenetubes.Theclearplastickind.Ifyoucan,buytheFalconbrandbecausetheyfittheinstrumentbest.Ifyoucan"t,don"tworry-wewillsupplythemwhenyoubringyoursamplestothelab.
- Icebucketwithcover.Generally,cellsaremorestableandtolerateinsultbetterwhenthey"recold.Theicebucketcoverkeepslightout,whichcouldbleachthefluorescentdye.
- Flowcytometer.Ifyouanalyzeyoursamplesinourlab,theinstrumentyouusewillmostlikelybeaFACScanorFACSort,madebyBecton-Dickinson.(Seeourinstrumentlistformoredetails.)
Procedure
Fixation
- Resuspendthecellsina500microlitervolumeofPBSandchillwellonice.
- Preparea12x75mm.tubecontaining500microlitersofice-cold100%ethanol.
- Rapidlypipetthecoldcellsuspensionintothecoldethanolandmixbyforcingairbubblesthroughthesuspension.Pasteur-typetransferpipetsworkbestforthis.
- Allowthesuspensiontoremainonicefor15minutes.
Thecellscanremaininthisstateindefinitelyifdesired,butwefindthatclumpingincreaseswiththedurationinalcohol.Ifitisnecessarytopausetheprocedurehere,makesurethetubesaresecurelysealedandkeptrefrigerated.
DebrisRemoval
- Carefullyunderlayerthecellsuspensionwith1mlice-coldcalfserumandcentrifuge3minutesat300xg.
- Carefullyaspiratetheliquid,takingcarenottodisturbthepellet.Usingacottonswab,carefullywipethesidesofthetubetoremoveanyattacheddebris.
- Add2ml.PBS.Vortex.
DNAStaining
- Centrifuge3minutesat300xg.RemoveasmuchliquidaspossIBLebutdonotdisturbthecellpellet,whichiseasilydetachedfromthetubewallafterethanolfixation.
- Add125microlitersofRNasesolution.Vortex.
- Incubatein37degreeC.waterbathfor15minutes.
- Removefromwaterbathandadd125microlitersofPIstainsolution.Vortex.
- Allowtostandatroomtemperatureforatleast30minutesbeforeanalyzingontheflowcytometer.
Thesamplesmayremainatroomtemperatureforupto2hours.Keepthecellsoniceorrefrigeratethemifitislongeruntilyourscheduledtimeontheflowcytometer.
Pipettingofthefinalcellsuspensionthroughanylonmonofilamentmeshscreenwith44micronopenings(wewillsupplytheseinourlab)immediatelypriortoanalysisontheflowcytometerishighlyrecommendedtoremovelargemulticellularaggregatescommoninethanol-fixedprepartions.
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