Overview

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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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