| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 492/650 |
| MW | ~1100 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | GPCR CalciumGPCRAssays |
| Related | CalciumChannels pHandIonIndicators BiochemicalAssays |
UseofCal-520®AM,Cal-590™AM,orCal-630™AMEsters
1.LoadCellswithCal-520®,Cal-590™orCal-630™AMEsters:
AMestersarenon-polarestersthatcanreadilycrosslivecellmembranes,andrapidlyhydrolyzedbycellularesterasesinsidelivecells.AMestersarewidelyusedforloadingavarietyofpolarfluorescentprobesintolivecellsnoninvasively.However,cautionsmustbeexercisedwhenAMestersareusedsincetheyaresusceptibletohydrolysis,particularlyinsolution.Theyshouldbereconstitutedjustbeforeuseinhigh-quality,anhydrousdimethylsulfoxide(DMSO).DMSOstocksolutionsmaybestoreddesiccatedat–20°Candprotectedfromlight.Undertheseconditions,AMestersshouldbestableforseveralmonths.FollowingisourrecommendedprotocolforloadingCal-520®AM,Cal-590™AMorCal-630™AM estersintolivecells.Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.
a) Preparea2to5mMstocksolutionofCal-520®AM,Cal-590™AMorCal-630™AMestersinhigh-quality,anhydrousDMSO.
b) Onthedayoftheexperiment,eitherdissolveCal-520®AM,Cal-590™AMorCal-630™AMinDMSOorthawanaliquotoftheindicatorstocksolutiontoroomtemperature.Prepareadyeworkingsolutionof10to20µMinHanksandHepesbuffer(HHBS)orthebufferofyourchoicewith0.04%Pluronic®F-127.Theexactconcentrationoftheindicatorrequiredforcellloadingmustbedeterminedempirically.
Note:ThenonionicdetergentPluronic®F-127issometimesusedtoincreasetheaqueoussolubilityofCal-520®AM,Cal-590™AMorCal-630™AMesters.AvarietyofPluronic®F-127solutionscanbepurchasedfromAATBioquest.
c) Ifyourcells(suchasCHOcells)containorganicanion-transports,thenprobenecid(1-2mM)maybeaddedtothedyeworkingsolution(finalinwellconcentrationwillbe0.5-1mM)toreduceleakageofthede-esterifiedindicators.
Note:AvarietyofReadiUse™probenecidincludingwatersolublesodiumsaltandstABIlizedsolutioncanbepurchasedfromAATBioquest
d) Addequalvolumeofthedyeworkingsolution(fromStepborc)intoyourcellplate.
e) Incubatethedye-loadingplateinacellincubatorfor60to90minutes,andthenincubatetheplateatroomtemperatureforanother30minutes.
Note:Incubatingthedyelongerthan2hoursgivesbettersignalintensityforsomecelllines.
f) ReplacethedyeworkingsolutionwithHHBSorifapplicable,abufferofyourchoicethatcontainsananiontransporterinhibitor,suchas1mMprobenecid,toremoveexcessprobes.
g) RuntheexperimentsatEx/Em=490/525nm(forCal-520®AM),540/590nm(forCal-590™AM)or600/640nm(forCal-630™AM).
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,andthecellswereincubatedat37oCfor2hours. ATP(50µL/well)wasaddedbyFlexStation(MolecularDevices)toachievethefinalindicatedconcentrations.
Cal590™AM Cal630™AM
Control ATP Control ATP
Figure3.ResponseofendogenousP2YreceptortoATPinCHO-Kcells.CHO-Kcellswereseededovernightat40,000cellsper100µLperwellina96-wellblackwall/clearbottomcostarplate.100µlof4µMCal590™AMorCal630™AMinHHBSwith1mMprobenecidwereaddedintothewells,andthecellswereincubatedat37°Cfor2hour.Thedyeloadingmediumswerereplacedwith100µlHHBSand1mMprobenecid,thenimagedwithafluorescencemicroscope(OlympusIX71)usingTRITCchannelbeforeandafteradding50µlof300µMATP.
| References&Citations |  PrinterFriendlyVersion |
- WendtER,FerryH,GreavesDR,KeshavS.(2015)Ratiometricanalysisoffuraredbyflowcytometry:atechniqueformonitoringintracellularcalciumfluxinprimarycellsubsets.PLoSOne,10,e0119532.
- BaileyS,MacardlePJ.(2006)AflowcytometriccomparisonofIndo-1tofluo-3andFuraRedexcitedwithlowpowerlasersfordetectingCa(2+)flux.JImmunolMethods,311,220.
- SuZL,LiN,SunYR,YangJ,WangIM,JiangSC.(1998)[Monitoringcalciuminouterhaircellswithconfocalmicroscopyandfluorescenceratiosoffluo-3andfura-red].ShiYanShengWuXueBao,31,323.
- WalczyskoP,WagnerE,AlbrechtovaJT.(2000)Useofco-loadedFluo-3andFuraRedfluorescentindicatorsforstudyingthecytosolicCa(2+)concentrationsdistributioninlivingplanttissue.CellCalcium,28,23.
- WuY,ClusinWT.(1997)Calciumtransientalternansinblood-perfusedischemichearts:observationswithfluorescentindicatorfurared.AmJPhysiol,273,H2161.
- BlackwoodAM,SagnellaGA,MarkanduND,MacGregorGA.(1997)ProblemsassociatedwithusingFura-2tomeasurefreeintracellularcalciumconcentrationsinhumanredbloodcells.JHumHypertens,11,601.
- FlotoRA,Mahaut-SmithMP,SomasundaramB,AllenJM.(1995)IgG-inducedCa2+oscillationsindifferentiatedU937cells;astudyusinglaserscanningconfocalmicroscopyandco-loadedfluo-3andfura-redfluorescentprobes.CellCalcium,18,377.
- NovakEJ,RabinovitchPS.(1994)Improvedsensitivityinflowcytometricintracellularionizedcalciummeasurementusingfluo-3/FuraRedfluorescenceratios.Cytometry,17,135.
- SchildD,JungA,SchultensHA.(1994)LocalizationofcalciumentrythroughcalciumchannelsinolfactoryreceptorneuronesusingalaserscanningmicroscopeandthecalciumindicatordyesFluo-3andFura-Red.CellCalcium,15,341.
- KurebayashiN,HarkinsAB,BaylorSM.(1993)Useoffuraredasanintracellularcalciumindicatorinfrogskeletalmusclefibers.BiophysJ,64,1934.
