| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 492/514 |
| MW | 1102.95 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | CellBIOLOGy pHandIonIndicators |
| Related |
- EnhancedBrightnessandSensitivity
- SignificantlyHigherSignaltoNoiseRatio
- MoreConsistentLoADIngandLocalization
- ConvenientSpectrumandEasyMultiplexing
TheHistory
Everysingleyear,therearehundredsofthousandsofpeer-reviewedpaperspublishedfocusingoncalciumanditsroleinbiologicalsystems.Thereasonforthisisclear.Calciumions(Ca2+)playacriticalpartinmanybiologicalprocesses,fromthecontractingofmusclestothereleaseofneurotransmitters.Thispervasivenessofcalciuminbiology,andthesubsequentneedtostudyit,haspushedforthedevelopmentofviable,convenientcalciumindicators,toolsthatresearchersmayusetoquantifyandvisualizeintracellularcalcium.
Thefirstbigbreakthroughcameinthe1980s,whenateamofscientistsattheUniversityofCaliforniaBerkeleydevelopedafluorescein-basedcalciumindicator.Theirresearchpavedthewayfornowwell-establishedcalciumprobessuchasFluo-3AMandFluo-4AM.Thesefluorescein-basedAMestersreadilypassthroughthemembranesoflivingcells,whereonceinside,theyarerapidlyhydrolyzedbyesterasesandsettodetectcalcium.Uponsuccessfulbindingwithcalcium,Fluo-3orFluo-4willbecomefluorescent,emittingasignalat~520nmwhenexcitedbya~490nmlightsource.
Asignificantachievementinitsownright,thesefirstgenerationfluorescein-baseddyesdidhoweversufferfromseveralmajordrawbacks.Oneofthesedrawbackswasthehighbackgroundfluorescence,sometimestermednoise.Ingeneral,noisecreatesproblemsindataanalysisandcanobscurepossIBLysignificantresults,andshouldbeminimizedasmuchaspossible.Thatiswhywesetouttosynthesizeanew,lownoisefluorescentcalciumindicator.Aftermuchresearchandoptimizingbyourdedicatedteamofexperts,wehavedevelopedanewhighsignaltonoiseratiocalciumindicator,Cal-520®AM.
Cal-520®AMFeatures
Cal-520®AMisanext-generationfluorescentcalciumindicatorthathassignificantimprovementsoverFluo-3AMandFluo-4AM.First,byimprovingthebindingspecificityofCal-520®forcalciumions,wehavemanagedtoproduceanindicatorwhichreducesautofluorescence.Thissimultaneouslylowersbackgroundfluorescenceandraisesthesignaltonoiseratio.ThebindingofCal-520®toCa2+willresultinagreaterthanonehundredfoldincreaseinfluorescenceintensity.Additionally,wehaveoptimizedCal-520®"schemicalstructuresothatitisbetterlocalized.UnlikeRhod-2,whichwilllocalizeinmitochondria,Cal-520®willpredominantlylocalizeinthecytosol.Lastly,wehavedevelopedCal-520®AMforeasyuseinmultiplexing.WhileCal-520®AMretainsthefamiliarspectraofFluo-4AMandFluo-8®AM,wealsohaveCal-590™AM(Ex/Em=573/588nm)andCal-630™(Ex/Em=608/626nm)forlongerwavelengthexcitations.
Table1.VariousExcitationsandEmissionsforMultiplexAssays
| CatNo. | Product | Ex(nm) | Em(nm) | Kd(nM) | AdditionalNotes |
| 21131 | Cal-520™AM | 492 | 514 | 320 | Bestusedwith488nmlaserandFITCfilterset |
| 20512 | Cal-590™AM | 573 | 588 | 561 | Bestusedwith555nmlaserandTRITC/Cy3filterset |
| 20532 | Cal-630™AM | 608 | 626 | 792 | Bestusedwith594nmlaserandTexasRed®filterset |
BecauseofCal-520®AM"sexcellentchemicalproperties,itisarobustprobeforfluorescence-basedassaydetectionofintracellularcalciummobilization.ItisperfectforevaluatingGPCRandcalciumchanneltargetsaswellasforscreeningtheiragoNISTsandantagonists.Additionally,Cal-520®AMiscompletelycompatiblewithuseinflowcytometry,fluorescencemicroscopy,fluorescencespectroscopyandthefluorescencemicroplateplatform.Forasamplingofspecificapplications,pleaseconsulttheReferencesandCitationssectionbelow.
| Spectrum | AdvancedSpectrumViewer |
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:
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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 |  CitationExplorer |
Below,youmayfindasmallsamplingofspecificCal-520®AMapplications.ToinquireaboutapotentialapplicationofCal-520®AM,ortoconsultwithourfluorescentdyespecialists,pleasecontactusatsupport@aatbio.comor1-800-990-8053.
InNeurobiology,Cal-520®AMhasbeenusedtostudy:
»Neuronsingleactionpotentialsinneocorticalneuronsbothinvitroandinvivo,andassociatedvoltage-dependentcalciumchannels[1]
»Superiorcolliculusinmiceinconjunctionwithtwo-photoncalciumimagingtovisualizeretinalganglioncellsinthesuperficiallamina[2]
»AldolaseCcompartmentsinmice,lookingatcomplexspikesynchronyanditsrelationtosensoryprocessinginawakeanimals[3]
»Neuralcircuitsinbrainsliceandwholebrainpreparation,specificallylookingatindividualactionpotentialsinvivo[4]
»Ca2+dependentcellsignalingpathwaysusingculturedhumanneuroblastomaSH-SY5Ycellsandhigh-speedvideo-microscopy[5]
InCellSignaling,Cal-520®AMhasbeenusedtostudy:
»Intracellularcalciuminspermusingthemicroplatereaderplatformasameanstoquantitatingparameterssuchasmotility[6]
»Calciumsignalingpathwaysinmeniscusfibrochondrocytesbywayofvisualizingcalciumlocalizationandconcentration[7]
»Ca2+mediatedcellularsignalinginrelationtoinositoltriphosphateanditsfluxthroughgapjunctions[8]
»Retinalwave-mediatedformationofcalciumtransientsinMüllerglialcellswithfocusonexpressionofGCaMP3[9]
»Ca2+signalingpathwaysinzebrafishsperm,specificallylookingatcalciumfluxresultingfromcGMP-inducedhyperpolarization[10]
InCardiology,Cal-520®AMhasbeenusedtostudy:
»Sarcoplasmicreticuluminsofarasitsroleincardiacexcitation-contractioncouplingandcalciumsparkevents[11]
»Opticalmappingofcalciumincardiactissueslicestodevelopaframeworkforfutureinvestigationsintocalciumtransients[12]
»Sodium-calciumexchangerfunctionalityandmechanismwithregardstoburstpacemakeractivityinknockoutmice[13]
»Pacemakermodulationinembryonicheartasafunctionofinositol-1,4,5-triphosphatereceptors[14]
»Calciumcurrentandtheroleofpotassiumchannel-interactingprotein2(KChIP2)inmicewithregardstoheartfailure[15]
