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AAT Bioquest/Cell Meter™ Fluorimetric Cell Cycle Assay Kit *Red Fluorescence Optimized for Flow Cytometry*/22842/100 T
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 535/617 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellAnalysis CellCytotoxicity |
| Related | ApoptosisandCytotoxicity CellApoptosis BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
1. Preparecells:
1.1. Treatcellswithtestcompoundsforadesiredperiodoftimetoinduceapoptosisorothercellcyclefunctions.
1.2. Foreachsample,preparecellsin0.5mLPBSatadensityof5×105to1×106cells/mL.
Note1:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityforapoptosisinduction.
Note2:ForAdherentCells:Thecellsaretrypsinized,sUSPendedin10%FBSmedium,centrifuged(1000rpm,5min),andthepelletsareresuspendedinPBS.
ForSuspensionCells:Thecellsarecentrifuged(1000rpm,5min),andthepelletssuspendedinPBS(1mL).
2. Fixthecellswith70%Ethanol:
Pipet0.5mLcellsuspension(fromStep1.2)into1.2mLabsoluteEthanol(finalconcentrationapprox.70%).Incubatecellsoniceforatleast2hours(orovernightat-20°C).Cellscanbestoredat-20°Cforupto2yearsbeforestaining.
Note1:Ethanoliscommonlyusedforfixationaftercellsurfaceantigenswerestainedwithmonoclonalantibodies,whilemethanoliscommonlyusedforfixationafterintracellularantigenswerestainedwithmonoclonalantibodies.
Note2:Inthisprocedurewholecellsarefixedandanalyzed.Becausetheentirecellmassisstillpresent,theuseofRNaseistypicallyincludedtoeliminateanydouble-strandedRNA.Despitethefactthatwholecellsarebeinganalyzed,attemptstodetectsomeintracellularantigensinconjunctionwithDNAmayfailbecausetheproteinsleakoutofthepermeabilizedcell(e.g.greenfluorescentprotein).Inthesecasesabriefpre-fixation(10minutesat4-6°C)with1%paraformaldehydeinPBSbeforethealcoholfixationwillhelpretaintheproteinsinthecell.
3. StainthecellswithNuclearRed™CCS1:
3.1. Pelletthecellsat1000rpmfor5minutes(fromStep2),andwashcellsatleastoncewithcoldPBS.
3.2. Suspendthepelletin0.5mLofAssaybuffer(ComponentC),andadd5μLof100XNuclearRed™CCS1(ComponentA)and5μLof100XRNaseA(ComponentB).Incubatethecellsatroomtemperaturefor30to60minutes.
Note:Theappropriateincubationtimedependsontheindividualcelltypeandcellconcentrationused.Optimizetheincubationtimeforeachexperiment.
3.3. Optional:Centrifugethecellsat1000rpmfor5minutes,andre-suspendcellsin0.5mLofassaybuffer(ComponentB)orbufferofyourchoice.
3.4. MonitorthefluorescenceintensitywithaflowcytometerusingtheFL2channel(Ex/Em=490/620nm).Gateonthecellsofinterest,excludingdebris.
| References&Citations |  PrinterFriendlyVersion |
2. EngstromJU,KmiecEB.(2008)DNAreplication,cellcycleprogressionandthetargetedgenerepairreaction.CellCycle,7,1402.
3. KuoHM,ChangLS,LinYL,LuHF,YangJS,LeeJH,ChungJG.(2007)MorininhibitsthegrowthofhumanleukemiaHL-60cellsviacellcyclearrestandinductionofapoptosisthroughmitochondriadependentpathway.AnticancerRes,27,395.
4. LouieMC,RevenkoAS,ZouJX,YaoJ,ChenHW.(2006)Directcontrolofcellcyclegeneexpressionbyproto-oncogeneproductACTR,anditsautoregulationunderliesitstransformingactivity.MolCellBiol,26,3810.
5. EaswaranHP,LeonhardtH,CardosoMC.(2005)CellcycleMarkersforlivecellanalyses.CellCycle,4,453.
6. EssersJ,vanCappellenWA,TheilAF,vanDrunenE,JaspersNG,HoeijmakersJH,WymanC,VermeulenW,KanaarR.(2005)Dynamicsofrelativechromosomepositionduringthecellcycle.MolBiolCell,16,769.
7. LunaL,RolsethV,HildrestrandGA,OtterleiM,DantzerF,BjorasM,SeebergE.(2005)DynamicrelocalizationofhOGG1duringthecellcycleisdisruptedincellsharbouringthehOGG1-Cys326polymorphicvariant.NucleicAcidsRes,33,1813.
8. Baran-MarszakF,FeuillardJ,NajjarI,LeClorennecC,BechetJM,Dusanter-FourtI,BornkammGW,RaphaelM,FagardR.(2004)DifferentialrolesofSTAT1alphaandSTAT1betainfludarabine-inducedcellcyclearrestandapoptosisinhumanBcells.Blood,104,2475.
9. ConlonKA,ZharkovDO,BerriosM.(2004)Cellcycleregulationofthemurine8oxoguanineDNAglycosylase(mOGG1):mOGG1associateswithmicrotubulesduringinterphaseandmitosis.DNARepair(Amst),3,1601.
10. NunezR,GarayN,VillafaneC,BrunoA,LindgrenV.(2004)DescriptionofaflowcytometryapproachbasedonSYBR-14stainingfortheassessmentofDNAcontent,cellcycleanalysis,andsortingoflivingnormalandneoplasticcells.ExpMolPathol,76,29.
