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Flow Cytometry Analysis
来自 : 蚂蚁淘

byQingMa,6/22/2000


Purpose

Flowcytometryemploysinstrumentationthatscanssinglecellsflowingpastexcitationsourcesinaliquidmedium.Thetechnologycanproviderapid,quantitative,multiparameteranalysesonsingleliving(ordead)cellsbasedonthemeasurementofvisIBLeandfluorescentlightemission.Flowcytometryisawidelyusedmethodforcharacterizingandseparatingindividualcells.Thisbasicprotocolfocuseson:measurefluorescenceintensityproducedbyfluorescent-labledantibodiesandligandsthatbindspecificcell-associatedmolecules.

Material

  • Stainingsolution:L-15medium(SigmacatalogL-99002)plus2.5%FCS(4oC)
  • Fluorescent-labeledantibodies
  • 5mlpolystyrenetube
  • 96wellV-bottomplate
  • Propidiumiodidesolution
  • Fixationsolution:2%paraformaldehydeinPBS(4oC)
  • Standardfluorescentbeads
  • BectonDickinsonFACScan
  • CELLQuestSoftware

Procedure

ImmunofluorescenceStaining

TheStainingprocedureiscarriedoutat4oCanddark.Appropriatecontrolsareneededtogetcorrectresults,suchasunstainedcontrol,isotypeantibodystainedcontrolandpositivecontroletc.

  1. MakesinglecellsUSPensioninL-15ataconcentrationof1-2x107cells/ml
  2. Add50ulcellsuspensionto5mlpolystyrenetube(or96wellV-bottompalte)
  3. Add50ulofappropriatelydilutedlabeledantibodytothecellsandmixgentlely
  4. Incubateat4oCindarkfor30minutes
  5. Washcellsbyadding2mlL-15andcentrifuge5minutesat1000rpm(300xg)For96wellplate,washcellsthreetimeswith150ulL-15
  6. Resuspendstainedcellpelletsin400ulL-15at4oCforflowcytomery
  7. Cellscanberesuspendedinfixationsolutionandstoredfor1week(optional)
  8. Add10ulpropidiumiodidepriortoanalysistodetectdeadcells(optional)
  9. CalibratetheFACScanbystandardfluorescentbeads(optional)
FlowCytometryAnalysis

Flowcytometersarecomplexinstrumentsthatrequireawell-trainedoperator.PriortousetheBectonDickinsonFACScan,takeatrainingcoursewithanexperienceduser.Followtherulespostedintheworkingareaforappropriatemaintenanceoftheinstrument.

  1. TurnontheFACScanpowerandwaituntiltheindicatorlightchangesfromNOTREADYtoSTANDBY.RestartthecomputertoconnectwiththeFACScan.
  2. Openyouraccountonthedesktopandusetheappropriatetemplateforaquiringdata(seereferenceformakingcorrectsetting).
  3. TurnfluidcontrolvalvetoRUNandplacesampletubeonFACScan
  4. AnalysisdatawithCELLQuestSoftware

References

  1. BectonDickinsonImmunocytometrySystems
  2. CurrentProtocolsinImmunology

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