Theprocedurepicksupattheendofthedirectorindirectstainingprocedures.Thecellsareexpectedtobein12x75mm.plasticculturetubes,onemillioncellspertube.

Itshouldnotbeusedwiththeproceduretolabeldeadcells.Fixedcellshaveapermeablemembrane-thedyewouldenterallthecells.

Materials

1. 2XParaformaldehydeStockSolution.
   • Add2g.paraformaldehydeto100ml.PBS+azide.
   • Heatto70degreescelciusinafumehood,orina56degreecelciuswaterbath,justuntiltheparaformaldehydegoesintosolution.
   • Allowtocooltoroomtemperature,thenadjusttopH7.4using0.1M.NaOHor0.1M.HCl,asneeded.
   • Storeat4degreescelcius.

2. 0.5%ParaformaldehydeWorkingSolution.Add10ml.ofthe2%StockSolutionto30ml.PBS+azide.Storeat4degreescelcius.Thissolutionisstableforupto1week.

3. Antibody-labeledcellsinPBS+azide.Theymayhavebeenlabeledusingeitherthedirectorindirectlabellingprocedures.Concentrationshouldbe1millioncellsin1ml.

   Equipment

   1. pHmeter.Thisisinvolvedinmakingtheparaformaldehydestocksolution.

   2. Balancewitharesolutionofatleast0.1g.Again,thisistomaketheparaformaldehydestocksolution.

   3. Onelitergraduatedcylinderorvolumetricflask.Ditto.

   4. Centrifuge.YoushouldknowhowtheRPMtranslatesintoG-force.

   5. Pipetintherangeof500to1000microliters(0.5-1.0ml.).

   6. Vortexmixer.Youcouldmixbytappingorshakingthetubes,butamixerwillgivemuchmorereproducibleresultsinmostcases.

   7. Refrigerator.Tostorethepreservedcells.

   Procedure

   1. Followingthelastwashstep,centrifugethecellsandremovetheliquid,asdescribedinthedirectorindirectantibodylabellingprocedure.

   2. Add0.5to1.0ml.ofcold0.5%paraformaldehydesolution.Vorteximmediately.

   3. StorethecellsUSPensionat4degreescelciusinthedark.

      Analyzethecellsontheflowcytometerwithinoneweek.

      ---

      Reference

      1. Lanier,L.L.,andWarner,N.L.:Paraformaldehydefixationofhematopoieticcellsforquantitativeflowcytometry(FACS)analysis.J.Immunol.Meth.,47:25,1981.

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