| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 490/525 |
| MW | N/A |
| CAS# | N/A |
| Solvent | N/A |
| Storage | F/D/L |
| Category | GPCR CalciumGPCRAssays |
| Related | CalciumChannels pHandIonIndicators |
| Spectrum | AdvancedSpectrumViewer |
- Preparecells:
- Foradherentcells:Platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfora96-wellplateor10,000to20,000cells/well/25µLfora384-wellplate.
- Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinCellgrowthmediumorHHBSat125,000to250,000cells/well/100µLfora96-wellpoly-Dlysineplateor30,000to60,000cells/well/25µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
Note:Eachcelllineshouldbeevaluatedontheindividualbasistodeterminetheoptimalcelldensityfortheintracellularcalciummobilization
- PrepareFluo-8E™AMdye-loadingsolution(foroneplate):
- MakeFluo-8E™AMstocksolution:Add10µLofDMSOintoonevialofFluo-8E™AM(ComponentA),andmixthemwell.
Note:10µLofFluo-8E™AMstocksolutionisenoughfor50assays(halfofthe96-wellplate).UnusedFluo-8E™AMstocksolutioncanbealiquotedandstoredat<-20=""°c=""for=""more=""than=""one=""month=""if=""the=""tubes=""are=""sealed=""tightly.=""protect=""from=""light=""and=""avoid=""repeated=""freeze-thaw=""> - Make1Xassaybuffer:Make1Xassaybufferbymixing9mLofHHBS(ComponentC)into10XPluronic®F127Plus(1mL,ComponentB),andmixthemwell.
Note:10mLof1Xassaybufferisenoughforoneplate.Aliquotandstoreunused1Xassaybufferat<-20=""°c.=""protect=""from=""light=""and=""avoid=""repeated=""freeze-thaw=""> - MakeFluo-8E™AMdye-loadingsolutionfor1/2cellplate:Add10µLofFluo-8E™AMDMSOstocksolution(fromStep2.1)into5mLof1Xassaybuffer(fromStep2.3),andmixthemwell.Thisworkingsolutionisstableforatleast2hoursatroomtemperature.
- MakeFluo-8E™AMstocksolution:Add10µLofDMSOintoonevialofFluo-8E™AM(ComponentA),andmixthemwell.
- Runcalciumassay:
- Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofFluo-8E™AMdye-loadingsolution(fromStep2.4)intothecellplate.Donotremovethegrowthmediumfromthecellplate.
- Incubatethedye-loadingplateinacellincubatorfor45-60minutes.
- Prepare5XagoNISTcompoundwithHHBSoryourdesiredbuffer.
- Add50µLofthepreparedagonistfromstep3.3andrunthecalciumfluxassayimmediatelybymonitoringthefluorescenceintensityatEx/Em=490/525nmwithbottomreadmode.
Note:Toachievethebestresults,itisimportanttoruntheassaywithin1minuteaftertheadditionoftheagonist.Itisalsoimportanttomakesurethetimebetweentheagonistadditionandthebeginningoftheactualreadingstaysconstantforallthesamples.
| 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.
