| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 530/590 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | GPCR CalciumGPCRAssays |
| Related | CalciumChannels pHandIonIndicators BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
Warning:Donotaddadditionalprobenecid.
1.PrepareCells:
1.1 Foradherentcells:Platecellsovernightinthegrowthmediumat40,000to80,000cells/well/100µLfora96-wellplateor10,000to20,000cells/well/25µLfora384-wellplate.
1.2 Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinRhod-4NWdye-loADIngsolution(seeStep2.4)at125,000to250,000cells/well/100µLfora96-wellpoly-Dlysineplateor30,000to60,000cells/well/25µLfora384-wellpoly-Dlysineplate.Centrifugetheplatesat800rpmfor2minuteswithbrakeoffpriortotheexperiments
Note:Eachcelllineshouldbeevaluatedonanindividualbasistodeterminetheoptimalcelldensityfortheintracellularcalciummobilization.
2.PrepareRhod-4NWdye-loadingsolution:
2.1 Thaw1vialofRhod-4NW(ComponentA),1bottleof10XPluronic®F127Plus(ComponentB)and1bottleofHHBS(ComponentC)atroomtemperaturebeforeuse.
2.2 MakeRhod-4NWstocksolution:Add100µLofDMSOintothevialofRhod-4NW(ComponentA),andmixthemwell.
Note:10µLofRhod-4NWstocksolutionisenoughforoneplate.UnusedRhod-4NWstocksolutioncanbealiquotedandstoredat<-20oCformorethanonemonthifthetubesaresealedtightly.Protectfromlightandavoidrepeatedfreeze-thawcycles.
2.3 Make1Xassaybuffer:
a).ForCat.#36331(10plateskit):make1Xassaybufferadding9mLofHHBS(ComponentC)intothebottleof10XPluronic®F127Plus,(1mL,ComponentB),andmixthemwell.
b).ForCat.#36332(100plateskit):make1Xassaybufferbyaddingthewholebottleof10XPluronic®F127Plus(10mL,ComponentB)into90mLofHHBSbuffer(notincludedinthekit),andmixthemwell.
Note:10mLof1Xassaybufferisenoughforoneplate.Aliquotandstoreunused1Xassaybufferat<‑20 oC.Protectfromlightandavoidrepeatedfreeze-thawcycles.
2.4 MakeRhod-4NWdye-loadingsolutionforonecellplatebyadding10µLofDMSOreconstitutedRhod-4NW(fromStep2.2)into10mLof1Xassaybuffer(fromStep2.3),andmixingthemwell.Thisworkingsolutionisstableforatleast2hoursatroomtemperature.
3.Runcalciumassay:
3.1 Removethegrowthmediumfromthecellplate.
Note1:Itisimportanttoremovethegrowthmediuminordertominimizebackgroundfluorescence,andcompoundinterferencewithserumorculturemedia.
Note2:Alternatively,growthecellsingrowthmediumwith0.5-1%FBStoavoidmediumremovalstep.Inthiscase,2XdyeloadingsolutioninHHBSbufferisneeded.[Weoffer2separatenowashcalciumassaykits(Cat.#36334andCat.#36335)forthosewhouse0.5-1%FBSingrowthmediumtoavoidthemediumremovalstep].
3.2 Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofRhod-4NWdye-loadingsolution(fromStep2.4)intothecellplates(fromStep3.1).
3.3 Incubatethedye-loadingplateinacellincubatorfor30minutes,andthenincubatetheplateatroomtemperatureforanother30minutes.
Note1:Iftheassayrequires37oC,performtheexperimentimmediatelywithoutfurtherroomtemperatureincubation.
Note2:Ifthecellscanfunctionwellatroomtemperatureforlongertime,incubatethecellplateatroomtemperaturefor1-2hours.
3.4 PreparethecompoundplatewithHHBSorthedesiredbuffer.
3.5 RunthecalciumfluxassaybymonitoringthefluorescenceintensityatEx/Em=540/590nm.
| References&Citations |  PrinterFriendlyVersion |
1. TerritoPR,HeilJ,BoseS,EvansFJ,BalabanRS.(2007)Fluorescenceabsorbanceinner-filterdecomposition:theroleofemissionshapeonestimatesoffreeCa(2+)usingRhod-2.ApplSpectrosc,61,138.
2. BednarB,CunninghamME,KissL,ChengG,McCauleyJA,LivertonNJ,KoblanKS.(2004)KineticcharacterizationofnovelNR2BantagoNISTsusingfluorescencedetectionofcalciumflux.JNeurosciMethods,137,247.
3. MartinVV,BeierleinM,MorganJL,RotheA,GeeKR.(2004)Novelfluo-4analogsforfluorescentcalciummeasurements.CellCalcium,36,509.
4. StammC,delNidoPJ.(2004)ProteinkinaseCandmyocardialcalciumhandlingduringischemiaandreperfusion:lessonslearnedusingRhod-2spectrofluorometry.ThoracCardiovascSurg,52,127.
5. StammC,FriehsI,ChoiYH,ZurakowskiD,McGowanFX,delNidoPJ.(2003)Cytosoliccalciumintheischemicrabbitheart:assessmentbypH-andtemperature-adjustedrhod-2spectrofluorometry.CardiovascRes,59,695.
6. DuC,MacGowanGA,FarkasDL,KoretskyAP.(2001)CalibrationofthecalciumdissociationconstantofRhod(2)intheperfusedmouseheartusingmanganesequenching.CellCalcium,29,217.
7. DuC,MacGowanGA,FarkasDL,KoretskyAP.(2001)Calciummeasurementsinperfusedmouseheart:quantitatingfluorescenceandabsorbanceofRhod-2byapplicationofphotonmigrationtheory.BiophysJ,80,549.
8. LannergrenJ,WesterbladH,BrutonJD.(2001)ChangesinmitochondrialCa2+detectedwithRhod-2insinglefrogandmouseskeletalmusclefibresduringandafterrepeatedtetaniccontractions.JMuscleResCellMotil,22,265.
9. MacGowanGA,DuC,GlontyV,SuhanJP,KoretskyAP,FarkasDL.(2001)Rhod-2basedmeasurementsofintracellularcalciumintheperfusedmouseheart:cellularandsubcellularlocalizationandresponsetopositiveinotropy.JBiomedOpt,6,23.
10. MurielMP,LambengN,DariosF,MichelPP,HirschEC,AgidY,RubergM.(2000)Mitochondrialfreecalciumlevels(Rhod-2fluorescence)andultrastructuralalterationsinneuronallydifferentiatedPC12cellsduringceramide-dependentcelldeath.JCompNeurol,426,297.
