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AAT Bioquest/Cell Meter™ Fluorimetric Intracellular pH Assay Kit/21180/1000 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 503/528 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | CellBIOLOGy pHandIonIndicators |
| Related | CellMetabolism BiochemicalAssays |
AssayProtocolforStandardCellLoad(OnePlate)
1.PrepareCells:
1.1 Foradherentcells,platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfor96-wellor10,000to20,000cells/well/25µLfor384-wellplates.
1.2 Fornon-adherentcells,centrifugethecellsfromtheculturemediumandthensUSPendthecellpelletsingrowthmediumat125,000to250,000cells/well/100µLfor96-wellor30,000to60,000cells/well/25µLfor384-wellpoly-Dlysineplates. Centrifugetheplatesat800rpmfor2minuteswithbreakoffpriortotheexperiments
Note: Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.PrepareRatioWorks™BCFL,AMdye-loadingsolution(for1plate):
2.1 ThawalltheComponentsatroomtemperaturebeforeuse.
2.2 MakeRatioWorks™BCFL,AMstocksolutionbyadding200µLDMSOintoComponentA(RatioWorks™BCFL,AM)andmixingthemwell.
Note:20µLofreconstitutedRatioWorks™BCFL,AMisenoughfor1plate,unusedRatioWorks™BCFL,AMcan bealiquotedandstoredat<-20oCforonemonthifthetubesaresealedtightly,avoidedlightandrepeatedfreeze-thawcycles.
2.3 Make1Xassaybufferbyadding1mLofComponentB(10XPluronicF127Plus)into9mLofComponentC(HHBS),mixthemwell.
Note:Forcellsthatrequireprobenecidforloading(e.g.CHOcells),dilute50mMReadiUse™probenecid(ComponentC)atconcentrationof1to5mM(prefer5mMforCHOcells).
2.4 MakeRatioWorks™BCFL,AMdye-loadingsolutionforonecellplatebyadding20µLofDMSOreconstitutedRatioWorks™BCFL,AM(fromStep2.2)into10mLof1Xassaybuffer(fromStep2.3),mixingthemwell.Thisworkingsolutionisstableforatleast2hoursatroomtemperature.
3. RunpHAssay
3.1 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)RatioWorks™BCFL,AMdye-loadingsolutionintothecellplate(fromStep2.4).
Note: ItisimportanttoreplacethegrowthmediumwithHHBSbuffer(100µL/wellfor96-wellplateor25µL/wellfor384-wellplatebeforedye-loading)ifyourcompoundsinterferewiththeserum.
3.2 Incubatethedye-loadingplateatcellincubatorfor30minutes,andthenincubatetheplateatroomtemperatureforanother30minutes.
Note:Iftheassayrequires37oC,performtheexperimentimmediatelywithoutfurtherroomtemperatureincubation.
3.3 PreparethecompoundplatesbyusingHHBSoryourdesiredbuffer.
3.4 RunthepHassaybymonitoringthefluorescenceatEx/Em=490/535nm(cutoffat515nm)or505/535nmand430/535nm(cutoffat515nm)forratiomeasurements.Thecompoundadditionis50µL/well(96-wellplate)or25µL/well(384-wellplate).
Note:Theassayshouldbecompletewithin3to5minaftercompoundaddition,howeveraminimumof8mindatacollectionarerecommendedforduringassaydevelopment.
AssayProtocolforAcid-Load(One96-wellPlate)
1.PrepareCells:
1.1 Foradherentcells,platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfor96-wellplates.
1.2 Fornon-adherentcells,centrifugethecellsfromtheculturemediumandthensuspendthecellpelletsingrowthmediumat125,000to250,000cells/well/100µLfor96-wellpoly-Dlysineplates.Centrifugetheplatesat800rpmfor2minuteswithbreakoffpriortotheexperiments
Note: Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensity.
2.PrepareRatioWorks™BCFL,AMdye-loadingsolution(for1plate):
2.1 ThawalltheComponentsatroomtemperaturebeforeuse.
2.2 MakeRatioWorks™BCFL,AMstocksolutionbyadding200µLDMSOintoComponentA(RatioWorks™BCFL,AM)andmixingthemwell.
Note:10µLofreconstitutedRatioWorks™BCFL,AMisenoughfor1plate,unusedRatioWorks™BCFL,AMcan bealiquotedandstoredat<-20oCforonemonthifthetubesaresealedtightly,avoidedlightandrepeatedfreeze-thawcycles.
2.3 Make1Xassaybufferbyadding1mLofComponentB(10XPluronicF127Plus)into4mLofComponentC(HHBS),mixthemwell.
Note:Forcellsthatrequireprobenecidforloading(e.g.CHOcells),dilute50mMReadiUse™probenecid(ComponentC)atconcentrationof0.5to2.5mM(prefer2.5mMforCHOcells).
2.4 MakeRatioWorks™BCFL,AMdye-loadingsolutionforonecellplatebyadding10µLofDMSOreconstitutedRatioWorks™BCFL,AM
(fromStep2.2)into5mLof1Xassaybuffer(fromStep2.3),mixingthemwell.Thisworkingsolutionisstableforatleast2hoursatroomtemperature.
3. RunpHAssay
3.1 Removethegrowthmediumfromthecellplate.
3.2 Add50µL/well/96-wellplateRatioWorks™BCFL,AMdye-loadingsolutionintothecellplate(fromStep2.4).
3.3 Incubatethedye-loadingplateatcellincubatorfor30minutes,andthenincubatetheplateatroomtemperatureforanother30minutes.
Note:Iftheassayrequires37oC,performtheexperimentimmediatelywithoutfurtherroomtemperatureincubation.
3.4 Add5µLof220mMNH4Clandthencentrifugetheplatesfor5seconds,andIncubate15minutesatroomtemperature.
Note: NH4ClsolutionshouldbepreparedfreshlyinHHBS(ComponentC).
3.5 PreparethecompoundplatesbyusingHHBSoryourdesiredbuffer.
3.6 RunthepHassaybymonitoringthefluorescenceatEx/Em=490/535nm(cutoffat515nm)or505/535nmand430/535nm(cutoffat515nm)forratiomeasurements.Thecompoundadditionis200µL/well/96-wellplate.
Note:Theassayshouldbecompletewithin3to5minaftercompoundaddition,howeveraminimumof8mindatacollectionarerecommendedforduringassaydevelopment.
| References&Citations |  CitationExplorer |
TheRedoxStatusofCancerCellsSupportsMechanismsbehindtheWarburgEffect
Authors:JorgelindodaVeigaMoreira,MinooHamraz,MohammadAbolhassani,ErwanBigan,SABInePérès,LoicPaulevé,MarcelLevyNogueira,Jean-MarcSteyaert,LaurentSchwartz
Journal:Metabolites(2016):33
TheRedoxStatusofCancerCellsSupportsMechanismsbehindtheWarburgEffect
Authors:JorgelindoDaVeigaMoreira,MinooHamraz,MohammadAbolhassani,ErwanBigan,SabinePérès,LoicPaulevé,MarcelLevyNogueira,Jean-MarcSteyaert,LaurentSchwartz
Journal:Metabolites(2016):33
