1. Add100mlofwell-mixedanticoagulatedwholebloodtothebottomofalabeledtube.
2. Addtheappropriateprimaryantibodytoeachtube.Ifusingunlabeledantibody,atitrationissuggested.Conjugatedantibodyshouldbeusedasdirected,usually20mlpersample.
3. Mixwell,thenincubateinthedarkatroomtemperaturefor20-30minutes.
4. Removetubesfromdarkchamberandmixeachtubewell.Add2mloflysingsolutiontoeachtube.Individuallyvortexeachtube.
5. Incubateatroomtemperatureinthedarkfor10-15minutes.
6. Centrifugefor5minutesat1000rpm(200xg).
7. Removesupernatantbyaspiration,vortex,andadd2mlswashingsolutiontoeachtube.
8. Centrifugefor5minutesat1000rpm(200xg).
9. Removesupernatantbyaspiration.
10. Ifrequired,addappropriatesecondstepantibody(atoptimalconcentration)toeachtubeandvortexgently(ifsecondstepantibodyisnotrequired,proceedtostep18).
11. Incubateinthedarkatroomtemperaturefor20-30minutes.
12. Removefromthedark.
13. Mixwell,thenadd2mlwashingbuffertoeachtube.
14. Centrifugefor5minutesat1000rpm(200xg).
15. Removesupernatantbyaspirationandvortex.
16. Ifthirdstepisnotrequired,proceedtostep18.Forthirdstep,addSav-PEtoeachtubeandvortexgently.
17. Repeatsteps11-15.
18. Ifyouwillanalyzethesameday,add500mlwashbuffertoeachtube,vortex,andanalyzewithin8hours.Ifnot,add2%formaldehydebuffertoeachtube,vortex,andstoreintherefrigeratorat2-8ºCforupto36hours.

Solutions:

WashingSolution:PBS+0.1%sodiumazide+1%fetalbovineserum.

Formaldehydebuffer:2%formaldehydeinPBS.

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