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Millipore/FCCS025100 | FlowCellect? PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection kit/FCCS025100/25 Test
  • Millipore/FCCS025100 | FlowCellect? PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection kit/FCCS025100/25 Test

Millipore/FCCS025100 | FlowCellect? PI3K/MAPK Dual Pathway Activation and Cancer Marker Detection kit/FCCS025100/25 Test

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貨號(hào): FCCS025100
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    • Description
      CatalogueNumberFCCS025100
      TradeName
      • FlowCellect
      DescriptionFlowCellect?PI3K/MAPKDualPathwayActivationandCancerMarkerDetectionkit
      OverviewRecentevidencesuggeststhatcross-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
      • FlowCellect
      BackgroundInformationExaminationofcellsignalingpathwaysandmonitoringtheiractivationstatushavebeenextremelyimportantforresearcherstounderstandthedetailedmechanismsofcellularfunctionsandthecauseofvariousdiseases.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.

      ProductInformation
      Components
      • 1.20XAnti-phospho-Erk1/2(Thr202/Tyr204,Thr185/Tyr187)R-PhycoerythrinconjugateMonoclonalAntibody:(PartNo.CS203329)One150μLvial
      • 2.20XAnti-phospho-Akt1/PKBα(Ser473)AlexaFluor488conjugateMonoclonalAntibody:(PartNo.CS203326)One150μLvial
      • 3.20XAnti-KI-67PerCPconjugateMonoclonalAntibody:(PartNo.CS203324)One150μLvial
      • 4.FixationBuffer:(PartNo.CS202122)One13mLbottle
      • 5.10XWashBuffer:(PartNo.CS202123)One13mLbottle
      • 6.5XAssayBuffer:(PartNo.CS202124)One55mLbottle
      • 7.1XPermeabilizationBuffer:(PartNo.CS202125)One13mLbottle
      • 8.CellCycleStop?:(PartNo.CS203335)Onevialsuppliedwith1mg
      Detectionmethod125IFluorescent
      StorageandShippingInformation
      StorageConditions4-8°Cforantibodiesandbuffers
      Applications
      ApplicationThisFlowCellectPI3K/MAPKDualActivation&CancerDetectionKitisdesignedtoexaminethiscross-talkinamulti-parametricfashionbyproviding3validatedbiomarkerstomeasurespecificcellsignalingeventsinflowcytometryapplications.
      KeyApplications
      • FlowCytometry
      BiologicalInformation
      HostMouse
      SpeciesReactivity
      • Human
      AntibodyTypeMonoclonalAntibody
      PhysicochemicalInformation
      Dimensions
      MaterialsInformation
      MaterialsInformation
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