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AAT Bioquest/Screen Quest™ 10X calcium assay buffer with Phenol Red Plus™/36301/10 Plates
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | None/None |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | GPCR CalciumGPCRAssays |
| Related | CalciumChannels pHandIonIndicators BiochemicalAssays |
1. Thawthebottleatroomtemperaturebeforeuse.
2. Make1XScreenQuestTMcalciumassaybuffer:Add1mLof10XScreenQuestTMcalciumassaybufferwithPhenolRedPlus™(Cat.#36301)to9mLofHHBS(1XHank’swith20mMHepesbuffer,pH7.0),andmixthemwell.
Note:10mLof1Xassaybufferisenoughforoneplate.Aliquotandstoreunused10Xassaybufferat<‑20 °C.Protectfromlightandavoidrepeatedfreeze-thawcycles.
3. Make2Xdye-loADIngsolutionforonecellplate:AddDMSOreconstitutedfluorescentcalciumdyes(suchasIndo-1AM,Fura-2AM,Fluo-3AM,Fluo-4AMandFluo-8AM,Rhod-2AMandRhod-4AM)into10mLof1XScreenQuestTMcalciumassaybuffer(fromStep2),tomakethefinalwelldyeconcentration2Xofthedesiredconcentration,andmixthemwell.Theworkingsolutionisstableforatleast2hoursatroomtemperature.
4. Tothemicroplatewelladd2Xdye-loadingsolution(fromStep3)whichisthesamevolumeasthecellculturemedium(e.g.,100uL/well/96-wellor25μL/well/384-well).
5. Incubatethecellsina37oC,5%CO2incubatorforabout1hours,orasdesired.
6. PreparethecompoundplatewithHHBSoryourdesiredbuffer.
7. Runthecalciumfluxassay.
| References&Citations |  PrinterFriendlyVersion |
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2. BausE,UrbainJ,LeoO,AndrisF.(1994)FlowcytometricmeasurementofcalciuminfluxinmurineTcellhybridsusingFluo-3andanorganic-aniontransportinhibitor.JImmunolMethods,173,41.
3. BednarB,CunninghamME,KissL,ChengG,McCauleyJA,LivertonNJ,KoblanKS.(2004)KineticcharacterizationofnovelNR2BantagoNISTsusingfluorescencedetectionofcalciumflux.JNeurosciMethods,137,247.
4. ChengH,SongLS,ShirokovaN,GonzalezA,LakattaEG,RiosE,SternMD.(1999)Amplitudedistributionofcalciumsparksinconfocalimages:theoryandstudieswithanautomaticdetectionmethod.BiophysJ,76,606.
5. doCeuMonteiroM,SansonettyF,GoncalvesMJ,O"ConnorJE.(1999)FlowcytometrickineticassayofcalciummobilizationinwholebloodplateletsusingFluo-3andCD41.Cytometry,35,302.
6. EberhardM,ErneP.(1989)Kineticsofcalciumbindingtofluo-3determinedbystopped-flowfluorescence.BiochemBiophysResCommun,163,309.
7. EscobarAL,CifuentesF,VergaraJL.(1995)DetectionofCa(2+)-transientselicitedbyflashphotolysisofDM-nitrophenwithafastcalciumindicator.FEBSLett,364,335.
8. FallonKM,ShacklockPS,TrewavasAJ.(1993)DetectioninVivoofVeryRapidRedLight-InducedCalcium-SensitiveProteinPhosphorylationinEtiolatedWheat(Triticumaestivum)LeafProtoplasts.PlantPhysiol,101,1039.
9. GreimersR,TrebakM,MoutschenM,JacobsN,BoniverJ.(1996)Improvedfour-colorflowcytometrymethodusingfluo-3andtripleimmunofluorescenceforanalysisofintracellularcalciumion([Ca2+]i)fluxesamongmouselymphnodeB-andT-lymphocytesubsets.Cytometry,23,205.
10. HagarAF,SpitzerJA.(1992)TheeffectofendotoxemiaonconcanavalinAinducedalterationsincytoplasmicfreecalciuminratspleencellsasdeterminedwithFluo-3.CellCalcium,13,123.
