| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 524/551 |
| MW | 880.07 |
| CAS# | N/A |
| Solvent | Water |
| Storage | F/D/L |
| Category | GPCR CalciumGPCRAssays |
| Related | CalciumChannels pHandIonIndicators BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
UseofRhod-4™AMEsters
1.LoadCellwithQuestRhod-4™AMEsters:
AMestersarethenon-polarestersthatreadilycrosslivecellmembranes,andrapidlyhydrolyzedbycellularesterasesinsidelivecells.AMestersarewidelyusedforloadingavarietyofpolarfluorescentprobesintolivecellnon-invasively.However,cautionsmustbeexcisedwhenAMestersareusedsincetheyaresusceptibletohydrolysis,particularlyinsolution.Theyshouldbereconstitutedjustbeforeuseinhigh-quality,anhydrousdimethylsulfoxide(DMSO).DMSOstocksolutionsmaybestoreddesiccatedat–20°Candprotectedfromlight.Undertheseconditions,AMestersshouldbestableforseveralmonths.
FollowingisourrecommendedprotocolforloadingRhod-4™AMestersintolivecells.Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.
a) Preparea2to5mMstocksolutionofRhod-4™AMestersinhigh-quality,anhydrousDMSO.
b) Onthedayoftheexperiment,eitherdissolveRhod-4™AMinDMSOorthawanaliquotoftheindicatorstocksolutiontoroomtemperature.Prepareaworkingsolutionof1to10µMinHanksandHepesbuffer(HHBS)orthebufferofyourchoicewith0.02%Pluronic®F-127.Formostofcelllines,Rhod-4™AMreagentswithaconcentrationrangingfrom4-5uMarerecommended.Theexactconcentrationoftheindicatorrequiredforcellloadingmustbedeterminedempirically.Toavoidanyartifactscausedbyoverloadingandpotentialdyetoxicity,itisrecommendedtousetheminimaldyeconcentrationthatcangeneratesufficientsignalstrength.
Note:ThenonionicdetergentPluronic®F-127issometimesusedtoincreasetheaqueoussolubilityofRhod-4™AMesters. AvarietyofPluronic®F-127solutionscanbepurchasedfromAATBioquest.
c) Ifyourcellscontainingtheorganicanion-transports,probenecid(1–2.5mM)orsulfinpyrazone(0.1–0.25mM)maybeaddedtothecellmediumtoreduceleakageofthede-esterifiedindicators.
Note:AvarietyofReadiUse™probenecidincludingwatersolublesodiumsaltandstABIlizedsolutioncanbepurchasedfromAATBioquest.
d) Addequalvolumeofthedyeworkingsolution(fromStepborc)intoyourcellplate.
e) Incubatethedye-loadingplateatacellincubatororroomtemperaturefor30minutestoonehouratroomtemperatureor37 °C.
Note:Decreasingtheloadingtemperaturemightreducethecompartmentalizationoftheindictor.
f) ReplacethedyeworkingsolutionwithHHBSorbufferofyourchoice(containingananiontransporterinhibitor,suchas2.5mMprobenecid,ifapplicable)toremoveexcessprobes.
g) RuntheexperimentsatEx/Em =540/590nm
2.MeasureIntracellularCalciumResponses:seefigure3.
TodetermineeitherthefreecalciumconcentrationofasolutionortheKdofasingle-wavelengthcalciumindicator,thefollowingequationisused:
[Ca]free=Kd[F─Fmin]/Fmax─F]
WhereFisthefluorescenceoftheindicatoratexperimentalcalciumlevels,FministhefluorescenceintheabsenceofcalciumandFmaxisthefluorescenceofthecalcium-saturatedprobe.
Thedissociationconstant(Kd)isameasureoftheaffinityoftheprobeforcalcium.TheCa-bindingandspectroscopicpropertiesoffluorescentindicatorsvaryquitesignificantlyincellularenvironmentscomparedtocalibrationsolutions.InsituresponsecalibrationsofintracellularindicatorstypicallyyieldKdvaluessignificantlyhigherthaninvitrodeterminations.InsitucalibrationsareperformedbyexposingloadedcellstocontrolledCa2+buffersinthepresenceofionophoressuchasA-23187,4-bromoA-23187andionomycin.Alternatively,cellpermeabilizationagentssuchasdigitoninorTriton®X-100canbeusedtoexposetheindicatortothecontrolledCa2+levelsoftheextracellularmedium.TheKdvalueofQuestRhod-4™islistedinTable1foryourreference.
UseofScreenQuest™Rhod-4NWCalciumAssayKitsforHTSApplications
GPCRactivationcanbedetectedbydirectmeasurementofthereceptormediatedcAMPaccumulation,orchangesinintracellularCa2+concentration.GPCRtargetsthatcoupleviaGqproduceanincreaseinintracellularCa2+thatcanbemeasuredusingacombinationofFluo-8®reagentsandafluorescencemicroplatereader.Thefluorescenceimagingplatereaders(suchas,FLIPR™,FDSSorBMGNovoStar™)haveacooledCCDcameraimagingsystemwhichcollectsthesignalfromeachwellofamicroplate(both96and384-well)simultaneously.Theseplatereaderscanreadatsub-secondintervals,whichenablesthekineticsoftheresponsetobecaptured,andhasanintegratedpipettorthatmaybeprogrammedforsuccessiveliquidadditions.BesidestheirrobustapplicationsforGPCRtargets,ourScreenQuest™Rhod-4CalciumAssayKitscanbealsousedforcharacterizingcalciumionchannelsandscreeningcalciumionchannel-targetedcompounds.
Rhod-4NW
EC50=1.2uM
Rhod-2AM
EC50=2uM
Figure3.CarbacholDoseResponsewasmeasuredinHEK-293cellswithScreenQuest™Rhod-4NWAssaykitandRhod-2AMunderthesameassayconditions.HEK-293cellswereseededovernightat40,000cells/100µL/wellina96-wellblackwall/clearbottomcostarplate.Thegrowthmediumwasremoved,andthecellswereincubatedwith100µLoftheScreenQuest™Rhod-4NWcalciumassaykitorRhod-2AM for1houratroomtemperature.Carbachol(25µL/well)wasaddedbyNOVOstar™(BMGLabTech)toachievethefinalindicatedconcentrations.TheEC50ofRhod-4NWisabout1.2uM.TheEx/Em=540/590nm.
OurScreenQuest™Rhod-4CalciumAssayKitshavethefollowingadvantagesforHTSapplications:
- LongerWavelengths:multipleexcitations@488,514,532&546nm;maximumemission@~555nm.
- NoWashRequiredandNoQuencherInterferencewithYourTargets.
- RobustPerformance:enablecalciumassaysthatareimpossiblewithRhod-2AM.
- LargerAssayWindow:10timeslargerthanRhod-2AM.
UseofRhod-4™Salts
Calciumcalibrationcanbecarriedoutbymeasuringthefluorescenceintensityofthesaltform(25to50µMinfluorescencemicroplatereaders)oftheindicatorsinsolutionswithpreciselyknownfreeCa2+concentrations.Calibrationsolutionscanbeusedbasedon30mMMOPSEGTACa2+buffer.Ingeneral,watercontainstraceamountofcalciumion.Itishighlyrecommendedtouse30mMMOPS+100mMKCl,pH7.2asbuffersystem.Onecansimplymakea0and39µMcalciumstocksolutionsaslistedbelow,andthese2solutionsareusedtomakeaserialsolutionofdifferentCa2+concentrations
A.0µMcalcium:30mMMOPS+100mMKCl,pH7.2buffer+10mMEGTA
B.39µMcalcium:30mMMOPS+100mMKCl,pH7.2buffer+10mMEGTA+10mMCaCl2
TodetermineeitherthefreecalciumconcentrationofasolutionortheKdofasingle-wavelengthcalciumindicator,thefollowingequationisused:
[Ca]free=Kd[F─Fmin]/Fmax─F]
WhereFisthefluorescenceintensityoftheindicatorataspecificexperimentalcalciumlevel,FministhefluorescenceintensityintheabsenceofcalciumandFmaxisthefluorescenceintensityofthecalcium-saturatedprobe.
Thedissociationconstant(Kd)isameasureoftheaffinityoftheprobeforcalcium.Thecalcium-bindingandspectroscopicpropertiesoffluorescentindicatorsvaryquitesignificantlyincellularenvironmentscomparedtocalibrationsolutions.InsituresponsecalibrationsofintracellularindicatorstypicallyyieldKdvaluessignificantlyhigherthaninvitrodeterminations.InsitucalibrationsareperformedbyexposingloadedcellstocontrolledCa2+buffersinthepresenceofionophoressuchasA-23187,4-bromoA-23187andionomycin.Alternatively,cellpermeabilizationagentssuchasdigitoninorTriton®X-100canbeusedtoexposetheindicatortothecontrolledCa2+levelsoftheextracellularmedium.TheKdvaluesofFluo-8®reagentsarelistedinTable1foryourreference.
| References&Citations |  CitationExplorer |
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Journal:Basicresearchincardiology(2017):44
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Journal:RSCAdvances(2016):35668--35676
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Journal:JournalofBiomedicalMaterialsResearchPartA(2016)
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Authors:RebekahAWarwick,MenachemHanani
Journal:Experimentalneurology(2016):10--18
MultiplepathwaysforelevatingextracellularadenosineintherathippocampalCA1regioncharacterizedbyadenosinesensorcells
Authors:KunihikoYamashiro,YukiFujii,ShoheiMaekawa,MitsuhiroMorita
Journal:JournalofNeuRochemistry(2016)
OptoDyCEasanautomatedsystemforhigh-throughputall-opticaldynamiccardiacelectrophysiology
Authors:AleksandraKlimas,ChristinaMAmbrosi,JinzhuYu,JohnCWilliams,HaroldBien,EmiliaEntcheva
Journal:Naturecommunications(2016)
TheGprotein-coupledreceptorGPR157regulatesneuronaldifferentiationofradialglialProgenitorsthroughtheGq-IP3pathway
Authors:YutakaTakeo,NobuhiroKurabayashi,MinhDangNguyen,KamonSanada
Journal:Scientificreports(2016)
