Materials

• Anti-cyclinD₁(Pharmingencat.no.14561A,cloneG12-4326)orcyclinD₂/D₃(cat.no.14711A,cloneG107-22).Othercyclinantibodiesmaybeappropriateaswell.

• Cy5-conjugatedanti-mouseIgG(JacksonImmunoResearchandCaltag)
• 0.5%paraformaldehydeinPBS
• 80%EtOHinddH₂0(storedat-20°C)
• cyclinstainingbuffer(1%BSAinPBSwith0.005%Tween20and0.1%sodiumazide)
• propidiumiodide(50µg/mlsolutionwith100U/mlDNase-freeRNase)
Procedure

• PreparethecelltypeofinterestasasinglecellsUSPensionandwashoncewithcoldPBSDecantthesupernatant,shaketubegentlytoresuspendpelletandadd1.0mlcold0.5%paraformaldehydeinPBS.Incubateforatleasttwohoursorovernightat4°C.
• WashthecellstwicewithcoldPBS/azideandplaceonice.Add2mls80%EtOHinddH₂0thathasbeenkept-20°C.Incubatethecellsforatleasttwohours.Washoncewithstainingbufferanddecant.
• Addtheprimaryanti-cyclinantibodyinavolumeof200µl.Incubateovernightat4°C.
• WashtwicewithcoldcyclinstainingbufferandaddtheCy5-conjugatedanti-mouseIgG(availablefromJacksonImmunoResearchandCaltag)inavolumeof200µl.Incubatefor4hoursat4°C
• WashoncewithcoldcyclinstainingbufferandoncewithcoldPBS/azide.Resuspendthecellsinpropidiumiodideat50µg/mlinPBSwith100U/mlRNase.Analyzeonany488nmargon-630nmHeNeordiodeduallaserflowcytometerforPIandCy5fluorescence.BothPIandCy5shouldbeanalyzedinlinearmode.

ThistechniquehasbeenusedsuccessfullytodetectcyclinD₁expressioninseveraltumorcelllines,activatedhumanlymphocytesandchickchondrocytes.Below,HL-60cellslabeledforcyclinD1orcyclinD2/D3with80%EtOHtreatmentat–20°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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