ThisprotocolisforusewiththeDandEcyclinsandemploys488nmargonlaserexcitationofpropidiumiodideandaFITC-conjugatedphenotypiclabel,and630nmNeNeordiodelaserexcitationoftheFluorochromeCy5todetectcellcycle-specificcyclinDexpression.Unlikepreviouslyusedbelow0°Cfixationprotocols,thismethodemploysagentleethanolfixationstep,preservingsurfaceMarkerexpressionandimmunolabeling.IthasbeentestedwithantibodiesagainstcyclinD₁(Pharmingencat.no.14561A,cloneG12-4326),cyclinD₂/D₃(cat.no.14711A,cloneG107-22)andcyclinE,andisbasedonprotocolsoriginallydesignedbyZ.Darzynkiewicz.Itcanalsobeusedwithothercyclinantibodies(suchasthoseagainstcyclinAandB₁)andantibodiesagainstproliferatingcellnuclearantigen(PCNA)showntobedetectablewithasingleethanolfixationstep.Thisassaycanbeusedwithanyinstrumentemployingduallaserexcitation,includingtheB-DVantage,FACSCaliburandCoulterElite.

Materials

• Anti-cyclinD₁(Pharmingencat.no.14561A,cloneG12-4326)orcyclinD₂/D₃(cat.no.14711A,cloneG107-22).Othercyclinantibodiesmaybeappropriateaswell.
• Cy5-conjugatedanti-mouseIgG(JacksonImmunoResearchandCaltag)
• FITC-conjugatedantibodyagainstsurfacemarkerofinterest0.1%paraformaldehydeinPBS
• 70%EtOHinglycerol(storedat4°C)
• stainingbuffer(1%BSAinPBSwith0.005%Tween20and0.1%sodiumazide)
• propidiumiodide(50mg/mlsolutionwith100U/mlDNase-freeRNase)
Procedure
• PreparethecelltypeofinterestasasinglecellsUSPensionandwashoncewithcoldPBSwith0.1%sodiumazide.LabelwiththeFITC-conjugatedantibodyofinterestincoldPBSwith2%FBSand0.1%sodiumazide.
• WashcellsoncewithPBS/FBS/azideandoncewithPBS/azidealone.Decantthesupernatant,shaketubegentlytoresuspendpelletandadd1.0mlcold0.1%paraformaldehydeinPBS.Incubateforatleasttwohoursorovernightat4oC.
• WashthecellstwicewithcoldPBS/azideandplaceonice.Add2mls70%EtOHinglycerolthathasbeenkeptat4°C.Incubatethecellsforatleasttwohours.Washoncewithstainingbufferanddecant.
• Addtheprimaryanti-cyclinantibodyinavolumeof200µl.Incubateovernightat4°C.
• WashtwicewithcoldstainingbufferandaddtheCy5-conjugatedanti-mouseIgG(availablefromJacksonImmunoResearchandCaltag)inavolumeof200µl.Incubatefor4hoursat4°C.
• WashoncewithcoldstainingbufferandoncewithcoldPBS/azide.Resuspendthecellsinpropidiumiodideat50µg/mlinPBSwith100U/mlRNase.Analyzeonany488nmargon-630nmHeNeordiodeduallaserflowcytometerforFITC,PIandCy5fluorescence.BothPIandCy5shouldbeanalyzedinlinearmode.

Thistechniquehasbeenusedsuccessfullytodetectcyclinexpressioninseveraltumorcelllines,activatedhumanlymphocytesandchickchondrocytes.Below,HL-60cellslabeledforcyclinD1eitherwith80%EtOHtreatmentat–20°Cor70%EtOHat4°C.

References

Gong,J.,Bhatia,U.,Traganos,F.andDarzynkiewicz,Z.1995.ExpressionofcyclinsA,D2andD3inindividualnormalmitogenstimulatedlymphocytesandinMOLT-4leukemiccellsanalyzedbymultiparameterflowcytometry.Leukemia9,893-899.

JuanG.,Gong,J.,Traganos,F.andDarzynkiewicz,Z.1996.UnscheduledexpressionofcyclinsD1andD3inhumantumourcelllines.CellProlif.29,259-266.

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