Whenstudyinginductionofapoptosisviaacellsurfacemolecule,itisimportanttofirstascertainsurfaceexpressionofthemoleculeofinterest.Thiscanbeachievedbyperformingflowcytometryorimmunoprecipitation/Westernblottingassays.Anotherfactorimportantinachievingoptimalresultsisthedegreeofsensitivityofcellstotheantibody-inducedapoptosis.Asanexample,CD95(Fas)-inducedapoptosisofhumanJurkatcelllinebymonoclonalantibodyEOS9.1isdescribedhereasageneralpositivecontrol.TheJurkatcellsfromdifferentsourcesusedinvariouslaboratoriesdemonstratelargevariABIlityintheirdegreeofsensitivitytotheFas-inducedapoptosis.Forbestresults,cellsshouldbeintheirexponentiallogphasewithviabilityrateofgreaterthan95%whentheyareusedforthisassay.

CD95(Fas)-inducedapoptosisofhumanJurkatcelllineusingEOS9.1mAb

  • Jurkatcells(cloneE6-1,ATCCCat.No.TIB-152)
  • RPMI-1640/10%FCS
  • Anti-humanCD95,cloneEOS9.1(eBioscienceCat.No.16-0958)
  • 24-wellcultureplate(CostarCat.No.3526)
  • 12x75mmflowcytometrycentrifugetubes(FalconCat.No.2008)
  • Apoptosisdetectionkit(eBioscienceApo-DirectCat.No.88-6611orApo-BrdUkitCat.No.88-6671)
    • Centrifuge
    • Pipettesandpipettors
    • Flowcytometer
      • 1/4hourforcellpreparation
      • Theincubationperiodisvariableandshouldbedeterminedempiricallybytheresearcher(asuggestedperiodtotryis16-18hours)
      • Prepareasingle-cellsUSPensionoftheJurkatcellline.Place5x105cellspermlofmediaina24-wellplateforeachsample.Use2-3wellsperconditiontoallowenoughcellsforsubsequentstainingandanalysis.
      • TreatcellsuspensionwithatitrationofEOS9.1mAb(arangeof1µg/mlto0.06µg/mlisrecommended).Cellsinwellstreatedwithmediumonlycanbeusedasunstimulatedcells.
      • Incubateovernight(16-18hoursoraslongasyourexperimentalquestionrequires).
      • Poolthecellsfromeachconditionandspincellsfor3-4minutesat300-400xg.
      • AspiratethesupernatantandfollowinstructionsoftheApo-DirectApoptosisDetectionKit.