PriortousinglymphoidtissuecellsUSPensionsforflowcytometricanalysisand/orforinvitrofunctionalassays,itisrecommendedtoremoveredbloodcells.TheeBioscience1XRBCLysisBufferisformulatedforoptimallysisoferythrocytesinsinglecellsuspensionsofmousetissuessuchasspleenandhumanperipheralblood.Thebuffercontainsammoniumchloride,whichlysesredcellswithminimaleffectonlymphocytes.Thisstepisnotnecessarywhenworkingwithmousethymusandlymphnodes.Whenusinghumanperipheralbloodforflowcytometricanalysis,aredcelllysingstepisincorporatedintothestainingprotocol.Forlysinghumanbloodinbulk,a10-20Xvolumeof1XRBClysisbuffertototalbloodisrecommended;however,analternativemethodforremovalofredbloodcellswhenworkingwithhumanbloodistouseisolationofPBMCbyFicoll-Hypaque™.

RBCLysisofMouseSpleen

• 1XPBS:
  • 80.0gNaCl80.0gNaCl
  • 11.6gNa₂HPO₄
  • 2.0gKH₂PO₄
  • 2.0gKCl
  • DIH₂0upto10.0L
  • pHto7.0
• eBioscience1XRBCLysisBuffer(Cat.No.00-4333)
• 50mlconicaltubes

• Pipettesandpipettors
• Centrifuge
• Hemacytometerandmicroscope

• 20minutes

1. Harvestmousespleenandprepareasinglecellsuspension.
2. Pelletthecellsbycentrifugation(300-400xg)at4°Candaspiratethesupernatant.
3. Resuspendthepelletin5ml/spleenofLysisBuffer.
4. Incubateatroomtemperaturefor4-5minuteswithoccasionalshaking(wehaveperformedthissteponicesuccessfullytoo).
5. StopthereactionbydilutingtheLysisBufferwith20-30mlof1XPBS.
6. Spinthecells(300-400xg)at4°Candresuspendthepelletintheappropriatebufferforuseinthenextstepofyourexperimentalprocedure.
7. Performacellcountatthistime.

Note:Ingeneral,asmallnumberofresidualredcellsdoesnotinterferewithproliferationassaysorcanbegatedoutfromflowcytometricanalysis.However,ifrequired,asecondroundoflysiscanbeperformed.