DescriptionThisprotocolisforusewiththeDcyclinsandemploys488nmargonlaserexcitationofpropidiumiodideand630nmNeNeordiodelaserexcitationoftheFluorochromeCy5todetectcellcycle-specificcyclinDexpression.IthasbeentestedwithantibodiesagainstcyclinD1(Pharmingencat.no.14561A,cloneG12-4326),cyclinD2/D3(cat.no.14711A,cloneG107-22)andcyclinEProcedurePreparethecelltypeofinterestasasinglecellsUSPensionandwashoncewithcoldPBSDecantthesupernatant,shaketubegentlytoresuspendpelletandadd1.0mlcold0.5%paraformaldehydeinPBS.Incubateforatleasttwohoursorovernightat4°C.WashthecellstwicewithcoldPBS/azideandplaceonice.Add2mls80%EtOHinddH20thathasbeenkept-20°C.Incubatethecellsforatleasttwohours.Washoncewithstainingbufferanddecant.Addtheprimaryanti-cyclinantibodyinavolumeof200µl.Incubateovernightat4°C.WashtwicewithcoldcyclinstainingbufferandaddtheCy5-conjugatedanti-mouseIgG(availablefromJacksonImmunoResearchandCaltag)inavolumeof200µl.Incubatefor4hoursat4°CWashoncewithcoldcyclinstainingbufferandoncewithcoldPBS/azide.Resuspendthecellsinpropidiumiodideat50µg/mlinPBSwith100U/mlRNase.Analyzeonany488nmargon-630nmHeNeordiodeduallaserflowcytometerforPIandCy5fluorescence.BothPIandCy5shouldbeanalyzedinlinearmode.RecipesAnti-cyclinD1(Pharmingencat.no.14561A,cloneG12-4326)orcyclinD2/D3(cat.no.14711A,cloneG107-22).Othercyclinantibodiesmaybeappropriateaswell.Cy5-conjugatedanti-mouseIgG(JacksonImmunoResearchandCaltag)0.5%paraformaldehydeinPBS80%EtOHinddH20(storedat-20°C)cyclinstainingbuffer(1%BSAinPBSwith0.005%Tween20and0.1%sodiumazide)propidiumiodide(50µg/mlsolutionwith100U/mlDNase-freeRNase)