AAT Bioquest/Cal-520FF™, AM/21142/1 mg

Overview

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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.

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.

Figure3.ResponseofendogenousP2YreceptortoATPinCHO-Kcells.CHO-Kcellswereseededovernightat40,000cellsper100µLperwellina96-wellblackwall/clearbottomcostarplate.100µlof4µMCal590™AMorCal630™AMinHHBSwith1mMprobenecidwereaddedintothewells,andthecellswereincubatedat37°Cfor2hour.Thedyeloadingmediumswerereplacedwith100µlHHBSand1mMprobenecid,thenimagedwithafluorescencemicroscope(OlympusIX71)usingTRITCchannelbeforeandafteradding50µlof300µMATP.

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