Immunodetection of cyclin D1 and D2/D3 using 488/630 nm dual laser flow cytometry相关交流-蚂蚁淘在线

Immunodetection of cyclin D1 and D2/D3 using 488/630 nm dual laser flow cytometry

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ImmunodetectionofcyclinD₁andD₂/D₃using488/630nmduallaserflowcytometry

WilliamTelfordHospitalforSpecialSurgery

ThisprotocolisforusewiththeDcyclinsandemploys488nmargonlaserexcitationofpropidiumiodideand630nmNeNeordiodelaserexcitationoftheFluorochromeCy5todetectcellcycle-specificcyclinDexpression.IthasbeentestedwithantibodiesagainstcyclinD₁(Pharmingencat.no.14561A,cloneG12-4326),cyclinD₂/D₃(cat.no.14711A,cloneG107-22)andcyclinE,andisbasedonprotocolsoriginallydesignedbyZ.Darzynkiewiczandcolleagues.AlthoughFITC-conjugatedsecondaryordirectFITC-conjugatedanti-cyclinantibodiescanalsobeused,thelowautofluorescencebackgroundseenwith630nmlaserexcitationmakesCy5detectionofcyclinexpressionparticularlysensitive.Usingduallaserexcitationalsoeliminatestheneedforfluorescencecompensation.Thisassaycanbeusedwithanyinstrumentemployingduallaserexcitation,includingtheB-DVantage,FACSCaliburandCoulterElite.

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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Immunodetection&nbsp;of&nbsp;cyclin&nbsp;D1&nbsp;and&nbsp;D2/D3&nbsp;using&nbsp;488/630&nbsp;nm&nbsp;dual&nbsp;laser&nbsp;flow&nbsp;cytometry

Immunodetection of cyclin D1 and D2/D3 using 488/630 nm dual laser flow cytometry