Overview

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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

OurCellMeter™assaykitsareasetoftoolsformonitoringcellviABIlityandproliferation.Thereareavarietyofparametersthatcanbeusedformonitoringcellviabilityandproliferation.Innormalcells,DNAdensitychangesdependingonwhetherthecellisgrowing,dividing,resting,orperformingitsordinaryfunctions.Theprogressionofthecellcycleiscontrolledbyacomplexinterplayamongvariouscellcycleregulators.Theseregulatorsactivatetranscriptionfactors,whichbindtoDNAandturnonorofftheproductionofproteinsthatresultincelldivision.Anymisstepinthisregulatorycascadecausesabnormalcellproliferationwhichunderliesmanypathologicalconditions,suchastumorformation.Potentialapplicationsforlive-cellstudiesareinthedeterminationofcellularDNAcontentandcellcycledistributionforthedetectionofvariationsingrowthpatterns,formonitoringapoptosis,andforevaluatingtumorcellbehaviorandsuppressorgenemechanisms.ThisparticularkitisdesignedtomonitorcellcycleprogressionandproliferationusingNuclearRed™CCS1,acellcyclestaininfixedcells.ThedyepassesthroughapermeabilizedmembraneandintercalatesintocellularDNA.ThesignalintensityofNuclearRed™CCS1isdirectlyproportionaltoDNAcontentafterRNAisdegradedbyRNaseprovidedinthekit.ThepercentageofcellsinagivensamplethatareinG0/G1,SandG2/Mphases,aswellasthecellsinthesub-G1phasepriortoapoptosiscanbemonitoredwithaflowcytometeratEx/Em=490nm/620nm(FL2channel).

Spectrum

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

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

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1.   FerulloDJ,CooperDL,MooreHR,LovettST.(2009)CellcyclesynchronizationofEscherichiacoliusingthestringentresponse,withfluorescencelabelingassaysforDNAcontentandreplication.Methods,48,8.

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.


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