Overview

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Ex/Em(nm)	458/597
MW	808.98
CAS#	N/A
Solvent	Water
Storage	F/D/L
Category	GPCR CalciumGPCRAssays
Related	CalciumChannels pHandIonIndicators BiochemicalAssays

FuraRedisavisIBLelight-excitablefura-2analogthatoffersuniquepossibilitiesforratiometricmeasurementofcalciumioninsinglecellsbymicrophotometry,imagingorflowcytometrywhenusedwithsingleexcitation,green-fluorescentcalciumindicators.FuraRedAMisthecell-permeableversionofFuraRedusedfornoninvasiveintracellularloADIng.FuraRedAMcanbesimultaneouslyloadedintocellswithFluo-3AM,Fluo-8AMorCal-520AM.Anadvantageofcombiningtwocalciumdyesisthatdyeswithlongerexcitationwavelengthscanbeused.ThisusuallycauseslessharmtothecellsthanusingratiometricdyesthatareexcitedwithUV-ornearUV-light(e.g.Fura-2),aslightatvisiblewavelengthsislessphototoxic.

Spectrum

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

UseofCalciumindicatorAMEsters

 

1.LoadCellswithCalciumIndicatorAMEsters:

AMestersarethenon-polarestersthatreadilycrosslivecellmembranes,andrapidlyhydrolyzedbycellularesterasesinsidelivecells.AMestersarewidelyusedforloadingavarietyofpolarfluorescentprobesintolivecellnon-invasively.However,cautionsmustbeexcisedwhenAMestersareusedsincetheyaresusceptibletohydrolysis,particularlyinsolution.Theyshouldbereconstitutedinhigh-quality,anhydrousdimethylsulfoxide(DMSO).DMSOstocksolutionsshouldbestoreddesiccatedat-20°Candprotectedfromlight.Undertheseconditions,AMestersshouldbestableforseveralmonths.

 

FollowingisourrecommendedprotocolforloadingAMestersintolivecells.Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

a)      Preparea2to5mMAMestersstocksolutioninhigh-quality,anhydrousDMSO.

b)      Onthedayoftheexperiment,eitherdissolvecalciumindicatorssolidinDMSOorthawanaliquotoftheindicatorstocksolutionstoroomtemperature.Prepareaworkingsolutionof2to20µMinthebufferofyourchoice(suchasHanksandHepesbuffer)with0.04%Pluronic®F-127.Formostcelllineswerecommendthefinalconcentrationofcalciumindicatorsbe4-5uM.Theexactconcentrationofindicatorsrequiredforcellloadingmustbedeterminedempirically.Toavoidanyartifactscausedbyoverloadingandpotentialdyetoxicity,itisrecommendedtousetheminimalprobeconcentrationthatcanyieldsufficientsignalstrength.

Note:ThenonionicdetergentPluronic®F-127issometimesusedtoincreasetheaqueoussolubilityofcalciumindicatorAMesters. AvarietyofPluronic®F-127solutionscanbepurchasedfromAATBioquest.

c)      Ifyourcells(suchasCHOcells)containingtheorganicanion-transports,probenecid(2–5mM)orsulfinpyrazone(0.2–0.5mM)maybeaddedtothethedyeworkingsolution(finalinwellconcentrationwillbe1-2.5mMforprobenecid,or0.1-0.25mMforsulfinpyrazone)toreducetheleakageofthede-esterifiedindicators.

Note:AvarietyofReadiUse™probenecidincludingwatersolublesodiumsaltandstABIlizedsolutioncanbepurchasedfromAATBioquest

d)      Addequalvolumeofthedyeworkingsolution(fromStepborc)intoyourcellplate.

e)      Incubatethedye-loadingplateroomattemperatureor37°Cfor20minutes(especiallyFluo-8AM)to2hours,andthenincubatetheplateatroomtemperatureforanother30minutes.

Note1:Decreasingtheloadingtemperaturemightreducethecompartmentalizationoftheindictor.

Note2:IncubatetheCal-520AMlongerthan2hoursgivesbettersignalintensityforsomecelllines.

f)       ReplacethedyeworkingsolutionwithHHBSorbufferofyourchoice(containingananiontransporterinhibitor,suchas1mMprobenecid,ifapplicable)toremoveexcessprobes.

g)      RuntheexperimentsatdesiredEx/Emwavelengths(seeTable1).

 

2.MeasureIntracellularCalciumResponses:

 

Figure1.ResponseofendogenousP2YreceptortoATPinCHO-M1cellswithoutprobenecid.CHO-M1cellswereseededovernightat40,000cellsper100µLperwellina96-wellblackwall/clearbottomcostarplate.100µlof4µMFluo-3AM,Fluo-4AMorCal520®AMinHHBSwereaddedintothewells,andthecellswereincubatedat37°Cfor2hour.Thedyeloadingmediumwerereplacedwith100µlHHBS,50µlof300µMATPwereadded,andthenimagedwithafluorescencemicroscope(OlympusIX71)usingFITCchannel.

A            B 

Figure2.ATP-stimulatedcalciumresponseofendogenousP2YreceptorinCHO-K1cellsmeasuredwithCal-520®orFluo-4AM.CHO-K1cellswereseededovernightin50,000cellsper100µLperwellina96-wellblackwall/clearbottomcostarplate.100µLof5µMFluo-4AMortheCal-520®AMwith(A)orwithout(B)2.5mMprobenecidwasaddedintothecells,andthecellswereincubatedat37^oCfor2hours. ATP(50µL/well)wasaddedbyFlexStation(MolecularDevices)toachievethefinalindicatedconcentrations.

 

UseofCalciumindicatorSalts

TodetermineeitherthefreecalciumconcentrationofasolutionortheK_dofasingle-wavelengthcalciumindicator,thefollowingequationisused:

[Ca]_free=K_d[F-F_min]/F_max-F]

WhereFisthefluorescenceoftheindicatoratexperimentalcalciumlevels,F_ministhefluorescenceintheabsenceofcalciumandF_maxisthefluorescenceofthecalcium-saturatedprobe.Thedissociationconstant(K_d)isameasureoftheaffinityoftheprobeforcalcium.TheCa²⁺-bindingandspectroscopicpropertiesoffluorescentindicatorsvaryquitesignificantlyincellularenvironmentscomparedtocalibrationsolutions.InsitucalibrationsofintracellularindicatorstypicallyyieldK_dvaluessignificantlyhigherthaninvitrodeterminations.InsitucalibrationsareperformedbyexposingloadedcellstocontrolledCa²⁺buffersinthepresenceofionophoressuchasA-23187,4-bromoA-23187andionomycin.Alternatively,cellpermeabilizationagentssuchasdigitoninorTriton®X-100canbeusedtoexposetheindicatortothecontrolledCa²⁺levelsoftheextracellularmedium.TheK_dvaluesofsomecalciumreagentsarelistedinTable1foryourreference.

 

UseofCalciumindicatorConjugates

 

Comparedtothefreeionindicator,dextranconjugatesofthesesameindicatorsexhibitbothreducedcompartmentalizationandmuchlowerratesofdyeleakage.Sincethemolecularweightofthedextran,netcharge,degreeoflabeling,andnatureofthedyemayaffecttheexperiment,researchersareadvisedtoconsulttheprimaryliteratureforinformationspecifictotheapplicationofinterest.

References&Citations

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1.   WendtER,FerryH,GreavesDR,KeshavS.(2015)Ratiometricanalysisoffuraredbyflowcytometry:atechniqueformonitoringintracellularcalciumfluxinprimarycellsubsets.PLoSOne,10,e0119532.

2.   BaileyS,MacardlePJ.(2006)AflowcytometriccomparisonofIndo-1tofluo-3andFuraRedexcitedwithlowpowerlasersfordetectingCa(2+)flux.JImmunolMethods,311,220.

3.   SuZL,LiN,SunYR,YangJ,WangIM,JiangSC.(1998)[Monitoringcalciuminouterhaircellswithconfocalmicroscopyandfluorescenceratiosoffluo-3andfura-red].ShiYanShengWuXueBao,31,323.

4.   WalczyskoP,WagnerE,AlbrechtovaJT.(2000)Useofco-loadedFluo-3andFuraRedfluorescentindicatorsforstudyingthecytosolicCa(2+)concentrationsdistributioninlivingplanttissue.CellCalcium,28,23.

5.   WuY,ClusinWT.(1997)Calciumtransientalternansinblood-perfusedischemichearts:observationswithfluorescentindicatorfurared.AmJPhysiol,273,H2161.

6.   BlackwoodAM,SagnellaGA,MarkanduND,MacGregorGA.(1997)ProblemsassociatedwithusingFura-2tomeasurefreeintracellularcalciumconcentrationsinhumanredbloodcells.JHumHypertens,11,601.

7.   FlotoRA,Mahaut-SmithMP,SomasundaramB,AllenJM.(1995)IgG-inducedCa2+oscillationsindifferentiatedU937cells;astudyusinglaserscanningconfocalmicroscopyandco-loadedfluo-3andfura-redfluorescentprobes.CellCalcium,18,377.

8.   NovakEJ,RabinovitchPS.(1994)Improvedsensitivityinflowcytometricintracellularionizedcalciummeasurementusingfluo-3/FuraRedfluorescenceratios.Cytometry,17,135.

9.   SchildD,JungA,SchultensHA.(1994)LocalizationofcalciumentrythroughcalciumchannelsinolfactoryreceptorneuronesusingalaserscanningmicroscopeandthecalciumindicatordyesFluo-3andFura-Red.CellCalcium,15,341.

10.   KurebayashiN,HarkinsAB,BaylorSM.(1993)Useoffuraredasanintracellularcalciumindicatorinfrogskeletalmusclefibers.BiophysJ,64,1934.

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