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AAT Bioquest/Screen Quest™ Luminometric Calcium Assay Kit/36305/10 Plates
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 437/466 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | GPCR CalciumGPCRAssays |
| Related | CalciumChannels pHandIonIndicators BiochemicalAssays |
1.Preparecells:
1.1 Foradherentcells:Platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfora96-wellplateor10,000to20,000cells/well/25µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinthecoelenterazine-loADIngsolution(seeStep2.4)at125,000to250,000cells/well/100µLfora96-wellpoly-Dlysineplateor30,000to60,000cells/well/25µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note:Eachcelllineshouldbeevaluatedontheindividualbasistodeterminetheoptimalcelldensityfortheintracellularcalciummobilization.
2.Preparecoelenterazine-loadingsolution:
2.1 Thawallthekitcomponentsatroomtemperaturebeforeuse.
2.2 Makecoelenterazineanalog:Add250µLof100%ETOH(ComponentB)intothevialofCoelenterazineAnalog(ComponentA),andmixthemwell.
Note:25µLofreconstitutedcoelenterazineanalogisenoughforoneplate.Unusedcoelenterazineanalogstocksolutioncanbestoredat<-20oCformorethanonemonthifthetubesaresealedtightly.Protectfromlightandavoidrepeatedfreeze-thawcycles.
2.3 Make1Xassaybuffer:
a)ForCat.#36305(10plateskit),readytouse1XAssayBuffer(ComponentC).
b)ForCat.#36306(100plateskit),make1Xassaybufferbydiluting10mLof10XAssayBuffer(ComponentC)into90mLofHHBSbuffer(notincludedinthekit),andmixthemwell.
Note:10mLof1Xassaybufferisenoughforoneplate.Storeunused1Xassaybufferat4oC.
2.4 Makecoelenterazine-loadingsolutionforonecellplate:Add25µLofETOHreconstitutedcoelenterazineanalog(fromStep2.2)into10mLof1Xassaybuffer(fromStep2.3),andmixthemwell.Thisworkingsolutionisstableforatleast2hoursatroomtemperature,protectedfromlight.
3.Runcalciumassay:
3.1 Removethegrowthmediumfromthecellplates.
Note1:Itisimportanttoremovethegrowthmediuminordertominimizecompoundinterferencewithserumorculturemedia.
Note2:Alternatively,growthecellsingrowthmediumwith0.5-1%FBStoavoidmediumremovalstep.Inthiscase,2Xcoelenterazine-loadingsolutionin1Xassaybufferisneeded.
3.2 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)coelenterazineloadingsolution(fromStep2.4)intothecellplates.
3.3 Incubatethecoelenterazine-loadingplatesatroomtemperaturefor3-4hours,protectedfromlight.
3.4 PreparethecompoundplateswithHHBSorthedesiredbuffer.
3.5 Monitortheaequorinluminescenceintensitybyusingthephotondetectionsystemthathasanenclosedchambercontainingaphotomultiplier.Theinstrumentmustcompletelyexcludeoutsidelight.
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