| CatalogueNumber | FCCS025100 |
| TradeName | |
| Description | FlowCellect™PI3K/MAPKDualPathwayActivationandCancerMarkerDetectionkit |
| Overview | Recentevidencesuggeststhatcross-talkbetweenthePI3KandMAPKsignalingpathwaysexist.WeusepAktandpERKantibodiestoexaminethePI3K/MAPKinteractions.Additionally,tofurtherinterrogatetheinterplaybetweenthesetwopathways,cellproliferativemarkerKi-67isusedtovalidatethefinalBIOLOGicaleffect.
Millipore’sFlowCellect™PI3K/MAPKDualActivationandCancerDetectionKitisdesignedtoexaminethiscross-talkinamulti-parametricfashionbyprovidingthreefullyvalidatedandoptimizedantibodybiomarkerstomeasurespecificcellsignalingeventsinflowapplications.ThethreeantibodiesprovidedinthekitareAnti-phospho-AktAlexaFluor488conjugate,Anti-phospho-ERKR-Phycoerythrinconjugate,andAnti-Ki-67PerCPconjugate.Byutilizingallthreeantibodybiomarkerssimultaneouslyinflowapplications,wenowhavetheABIlitytothoroughlyevaluatethe“cross-talk”betweenPI3KandMAPKpathwaysandtofurtherdeterminetheconsequenceoftheirinterplayincellproliferationanddifferentiationbymeasuringtheireffectonKi-67expression.
PhosphorylatedAktandphosphorylatedERKareincludedinthekittoprovidetheenduserwiththemeanstocross-examineboththePI3KandMAPKsignalingpathwayssimultaneously.IthasbeensuggestedthatthephosphorylationofAktcanresultintheinhibition,ordephosphorylation,ofphospho-RafonSer259[Jun,T.etal.(1999)].ByinactivatingRaf,thiswillessentiallyblocktheMAPKsignalingpathwayresultinginaninactivatedphospho-ERK.Insomesituations,asurfacereceptor(suchasIGF-1)willactivateboththePI3KandMAPKpathwaysleADIngtothephosphorylationofbothAktandERK.However,sincethisinteractionor“cross-talk”existbetweenthetwopathways,itiscriticaltoinvestigatetheirinteractionsinbothaspatialandtemporalmanner.
Inarecentstudy,wehaveexaminedtheeffectsofIGF-1activationbytheadditionofInsulinonboththePI3KandMAPKsignalingpathwaysonHEK293cells.Wehaveperformedthisexperimentimplementingcriticaltimepointsforsignalingevaluation:activationat3minuteandat5minutetimeintervals.Theresultingresponsesindicatethatcross-talkisobserved,notedbyasharpdecreaseinERKexpression.ThistransientresponseisattributedtothephosphorylationofAkt,whichinturnwillshutoffphosphorylatedERKasnotedabove.
Inordertovalidatethebiologicaleffectofphospho-proteinactivationbyagivenstimulus,cellcyclemarkerKi-67isusedsinceitisatruemeasurementofthe“proliferativefraction”.Ki-67ispresentinallphasesofthecellcycleexceptforG0.However,Ki-67canonlybedetectedinflowcytometrywhencellsaregoingthroughMphaseasKi-67expressionpatternsarepunctateinallotherphasesproducingweakersignals.Butbyusingacellcyclearrestreagent,CellCycleStop™,cellproliferationmeasuredbyKi-67expressioncanbeaccuratelydeterminedascellsarearrestedatMphaseandareclearlyvisIBLeinflowcytometryanalysis.InordertoaccuratelymeasurethecellproliferativeactivitybyKi-67expression,however,cellsmustbetreatedforatleast12hourswithacombinationofCellCycleStop™andagivencellstimulustofullyachieveenoughcirculatingcellstobecapturedinM-phase.Additionally,priortocelltreatmentsallculturesmustbeserumstarvedfor24hourstoessentiallyresetthecellcycleandbringmostcirculatingcellsbacktoG0[Littleton,RJ.etal.(1991)].
Usingmulti-parametricflowanalysis,weareabletocross-examinethesesignalingeventsandtheirbiologicalconsequencesimultaneously,providingabiologicalcorrelationbetweenpathwayactivationandcancerproliferation. |
| AlternateNames | |
| BackgroundInformation | Examinationofcellsignalingpathwaysandmonitoringtheiractivationstatushavebeenextremelyimportantforresearcherstounderstandthedetailedmechanismsofcellularfunctionsandthecauseofvariousdiseases.Manysignaltransductionpathwayshavebeenimplicatedtoleadtomultipleoutcomessuchasapoptosis,celldifferentiation,cellgrowthandcellproliferation,allofwhichhavebeenextensivelystudiedforthetreatmentofvariouscancersandautoimmunediseases.
Thestudyofcellsignalingpathwaysarenowmadeeasierwiththeuseofactivationstatus-specificandphospho-specificantibodies.Measurementofproteinphosphorylationwithphospho-specificantibodieshasgiveninsightintokinasesignalingcascades[Krutzik,P.O.etal.(2003)].Multi-parameterphosphoflowcytometryisapowerfultoolforstudyingmultiplepathwaysinamixedcellpopulationatthesametime.
Muchexcitementinthefieldofsignaltransductionhascenteredonthediscoveryofincreasingcross-talkamongsignalingpathways[Jun,T.etal.(1999)].RecentevidencehassuggestedthatcommunicationbetweenthePI3KandMAPKpathwaysexistdownstreamfromthecellsurface[Jun,T.etal.(1999),Moelling,K.etal.(2002),Zimmermann,S.etal.(1999)].Theabilityforsignalingpathwaystocross-talkaddsanextradimensionandcomplexitywhenevaluatingpathwaysofinterest.Sincesignaltransductionpathwaysareanelaboratehighwayofevents,theabilitytomonitorthesekeyintracellular“checkpoints”simultaneouslyprovidesresearchersaverypowerfultoolforanalyzingcomplicatedcelleventssuchascancercellproliferationbymeasuringtheactivityofmultiplecellsignalingpathways.
Millipore’sFlowCellect™PI3K/MAPKDualPathwayActivationandCancerMarkerDetectionkitisdesignedtoallowtheresearchertocrossexamineboththePI3KandMAPKsignalingpathways,aswellascellproliferationsimultaneously.Thiskitprovidesthreedirectlyconjugatedantibodieswhichareoptimizedformulti-colorflowcytometryapplicationsforthedetectionofAktphosphorylation,ERK1/2phosphorylationandKi-67cancermarkerexpression.Ki-67hasbeenindicatedtobeareliabletumorproliferativemarkerincancercells[Ishikuro,A.etal.(1997)].ThefractionofKi-67positivecells,oftendefinedasthe“proliferativefraction”,hasprognosticvalueinmanytumors[Darzynkiewicz,Z.etal.(2001)].
Althoughtheuseofphospho-specificantibodystainingasabiomarkermaygivesomemeasureoftargetactivation,itmaynotnecessarilycorrelatewiththedesiredbiologicaleffect(e.g.growthinhibitionorapoptosis).Researcherswouldlikelybenefitfromtheinclusionofbothphospho-specificsignaltransductionmarkers(suchaspERKandpAkt)andaproliferationmarker(suchasKi-67)toallowatruemeasureoftreatmentevaluationatthelevelofthetumor[Smalley,KSMetal.(2007)].
AllFlowCellect™kitsareoptimizedonthebench-topGuava®flowcytometrysystems,whichsavesvaluabletimeandsamplevolume.Allkitscontainoptimizedfixation,permeabilization,washandflowbufferstoprovideresearcherswithacompletesolutionforsimultaneousdetectionofmultiplepathwayactivations.WiththeGuavaplatformandFlowCellect™kits,onecanfinallyhaveaneasy,reliableandfullyvalidatedsolutiontostudythecomplexcellsignalingpathwaysrightinthecomfortofyourownlab.
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