立克次氏体通用探针法荧光定量PCR试剂盒厂家品牌:邦景进口、...
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
ImmunofluorescenceStainingTheStainingprocedureiscarriedoutat4oCanddark.Appropriatecontrolsareneededtogetcorrectresults,suchasunstainedcontrol,isotypeantibodystainedcontrolandpositivecontroletc.
- MakesinglecellsUSPensioninL-15ataconcentrationof1-2x107cells/ml
- Add50ulcellsuspensionto5mlpolystyrenetube(or96wellV-bottompalte)
- Add50ulofappropriatelydilutedlabeledantibodytothecellsandmixgentlely
- Incubateat4oCindarkfor30minutes
- Washcellsbyadding2mlL-15andcentrifuge5minutesat1000rpm(300xg)For96wellplate,washcellsthreetimeswith150ulL-15
- Resuspendstainedcellpelletsin400ulL-15at4oCforflowcytomery
- Cellscanberesuspendedinfixationsolutionandstoredfor1week(optional)
- Add10ulpropidiumiodidepriortoanalysistodetectdeadcells(optional)
- CalibratetheFACScanbystandardfluorescentbeads(optional)
FlowCytometryAnalysisFlowcytometersarecomplexinstrumentsthatrequireawell-trainedoperator.PriortousetheBectonDickinsonFACScan,takeatrainingcoursewithanexperienceduser.Followtherulespostedintheworkingareaforappropriatemaintenanceoftheinstrument.
- TurnontheFACScanpowerandwaituntiltheindicatorlightchangesfromNOTREADYtoSTANDBY.RestartthecomputertoconnectwiththeFACScan.
- Openyouraccountonthedesktopandusetheappropriatetemplateforaquiringdata(seereferenceformakingcorrectsetting).
- TurnfluidcontrolvalvetoRUNandplacesampletubeonFACScan
- AnalysisdatawithCELLQuestSoftware
References
- BectonDickinsonImmunocytometrySystems
- CurrentProtocolsinImmunology
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