Generally,themethodcanbecombinedwiththeproceduresfordirectlabelingorindirectlabelingofcellsurfaceantigens.UseonlyFITC-labeledantibodiessincePIemitsintheorangetoredregionofthespectrum(FITCemitsgreen).Anddonotfixthecellswithparaformaldehyde-startwiththisprotocolafterthelastwashstepinthedirectorindirectimmunofluorescenceprotocols.

Thefluorescencefrommanyantibody-labeledcellsurfaceantigensisoftenpartiallydiminishedfollowingthisprocedure.Otherantigensmaybetotallydenaturedandallfluorescencemaybelost.Itisbesttotestforthisbeforebeginningalargedual-staining(cellsurfaceandDNA)experiment.

Materials

1. DNAStain:5milligramsofPIin100millilitersof1.12%(w/v)ofsodiumcitrate.

2. RNAsesolution:500unitsperml.in1.12%(w/v)ofsodiumcitrate.

   UseahighlypurifiedRNAsetoavoiddestructionfothecellsbycontaminatingproteasesand/orDNAses.Otherwise,theRNasesolutioncanbepurifiedbyheatingto75degreescelciusfor30minutes.Cooltoroomtemperaturebeforetreatingthecells.

3. 100%ethanol.

4. Fetalornewbornbovineserum,(optional)forremovalofdebris.

5. CellsinsUSPension,countedandviability-checked.Theprocedureiswrittenforaliquotsof1millioncells,butyoucanscaleitupasrequired.ThecellsshouldbesuspendedinculturemediaorPBSwithoutserum,astheethanolfixationstepwillpercipitatetheproteinsintheserum.

   Thisproceduredoesnotnecessarilyrequire100%viablecells,butgenerallythemoredeadcellsyouhave,themoredebrisandmulti-cellaggregatesyoucanexpect.

   Equipment

   1. Centrifuge.YoushouldknowhowtheRPMtranslatesintoG-force.

   2. Precisionadjustablemicropipet.Itshouldcovertherangefrom100to1000microliters.

   3. Pasteur-typetransferPipettes,eitherglassorplastic.

   4. Cottonswabs.

   5. Vortexmixer.

   6. 37degreeC.waterbath.

   7. 12x75mmpolystyrenetubes.Theclearplastickind.Ifyoucan,buytheFalconbrandbecausetheyfittheinstrumentbest.Ifyoucan"t,don"tworry-wewillsupplythemwhenyoubringyoursamplestothelab.

   8. Icebucketwithcover.Generally,cellsaremorestableandtolerateinsultbetterwhenthey"recold.Theicebucketcoverkeepslightout,whichcouldbleachthefluorescentdye.

   9. Flowcytometer.Ifyouanalyzeyoursamplesinourlab,theinstrumentyouusewillmostlikelybeaFACScanorFACSort,madebyBecton-Dickinson.(Seeourinstrumentlistformoredetails.)

      Procedure

      Fixation

      1. Resuspendthecellsina500microlitervolumeofPBSandchillwellonice.

      2. Preparea12x75mm.tubecontaining500microlitersofice-cold100%ethanol.

      3. Rapidlypipetthecoldcellsuspensionintothecoldethanolandmixbyforcingairbubblesthroughthesuspension.Pasteur-typetransferpipetsworkbestforthis.

      4. Allowthesuspensiontoremainonicefor15minutes.

         Thecellscanremaininthisstateindefinitelyifdesired,butwefindthatclumpingincreaseswiththedurationinalcohol.Ifitisnecessarytopausetheprocedurehere,makesurethetubesaresecurelysealedandkeptrefrigerated.

         DebrisRemoval

      5. Carefullyunderlayerthecellsuspensionwith1mlice-coldcalfserumandcentrifuge3minutesat300xg.

      6. Carefullyaspiratetheliquid,takingcarenottodisturbthepellet.Usingacottonswab,carefullywipethesidesofthetubetoremoveanyattacheddebris.

      7. Add2ml.PBS.Vortex.

         DNAStaining

      8. Centrifuge3minutesat300xg.RemoveasmuchliquidaspossIBLebutdonotdisturbthecellpellet,whichiseasilydetachedfromthetubewallafterethanolfixation.

      9. Add125microlitersofRNasesolution.Vortex.

      10. Incubatein37degreeC.waterbathfor15minutes.

      11. Removefromwaterbathandadd125microlitersofPIstainsolution.Vortex.

      12. Allowtostandatroomtemperatureforatleast30minutesbeforeanalyzingontheflowcytometer.

          Thesamplesmayremainatroomtemperatureforupto2hours.Keepthecellsoniceorrefrigeratethemifitislongeruntilyourscheduledtimeontheflowcytometer.

          Pipettingofthefinalcellsuspensionthroughanylonmonofilamentmeshscreenwith44micronopenings(wewillsupplytheseinourlab)immediatelypriortoanalysisontheflowcytometerishighlyrecommendedtoremovelargemulticellularaggregatescommoninethanol-fixedprepartions.

          ---

          References

          Thismethodisbasedon,butmodifiedfromthefollowing:
          1. Braylan,RC,Benson,NA,Nourse,V,andKruth,HS:CorrelatedanalysisofcellularDNA,membraneantigens,andlightscatterofhumanlymphoidcells.Cytometry,2(5):337-343,1982.

          2. Crissman,HA,andSteinkamp,JA:RapidsimultaneousmeasurementofDNA,protein,andcellvolumeinsinglecellsfromlargemammaliancellpopulations.J.CellBiol.,59:766,1973.

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