Paraformaldehyde Fixation of Cells
来自 : 蚂蚁淘
ParaformaldehydeFixationofCells
Background
ThisfixationmethodisgoodforcellslabelledbyFluorochrome-conjugatedantibodiestomembraneantigens.ItwillstABIlizethelightscatterandlabellingforuptotoaweekinmostinstances,allowingyoutobemoreflexIBLeinschedulingcytometertime.FurThermore,itinactivatesmostbiohazardousagents,soitisimportantfromasafetystandpointaswell.Theprocedurepicksupattheendofthedirectorindirectstainingprocedures.Thecellsareexpectedtobein12x75mm.plasticculturetubes,onemillioncellspertube.
Itshouldnotbeusedwiththeproceduretolabeldeadcells.Fixedcellshaveapermeablemembrane-thedyewouldenterallthecells.
Materials
- 2XParaformaldehydeStockSolution.
- Add2g.paraformaldehydeto100ml.PBS+azide.
- Heatto70degreescelciusinafumehood,orina56degreecelciuswaterbath,justuntiltheparaformaldehydegoesintosolution.
- Allowtocooltoroomtemperature,thenadjusttopH7.4using0.1M.NaOHor0.1M.HCl,asneeded.
- Storeat4degreescelcius.
- 0.5%ParaformaldehydeWorkingSolution.Add10ml.ofthe2%StockSolutionto30ml.PBS+azide.Storeat4degreescelcius.Thissolutionisstableforupto1week.
- Antibody-labeledcellsinPBS+azide.Theymayhavebeenlabeledusingeitherthedirectorindirectlabellingprocedures.Concentrationshouldbe1millioncellsin1ml.
Equipment
- pHmeter.Thisisinvolvedinmakingtheparaformaldehydestocksolution.
- Balancewitharesolutionofatleast0.1g.Again,thisistomaketheparaformaldehydestocksolution.
- Onelitergraduatedcylinderorvolumetricflask.Ditto.
- Centrifuge.YoushouldknowhowtheRPMtranslatesintoG-force.
- Pipetintherangeof500to1000microliters(0.5-1.0ml.).
- Vortexmixer.Youcouldmixbytappingorshakingthetubes,butamixerwillgivemuchmorereproducibleresultsinmostcases.
- Refrigerator.Tostorethepreservedcells.
Procedure
- Followingthelastwashstep,centrifugethecellsandremovetheliquid,asdescribedinthedirectorindirectantibodylabellingprocedure.
- Add0.5to1.0ml.ofcold0.5%paraformaldehydesolution.Vorteximmediately.
- StorethecellsUSPensionat4degreescelciusinthedark.
Analyzethecellsontheflowcytometerwithinoneweek.
Reference
- Lanier,L.L.,andWarner,N.L.:Paraformaldehydefixationofhematopoieticcellsforquantitativeflowcytometry(FACS)analysis.J.Immunol.Meth.,47:25,1981.
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