| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 573/588 |
| MW | 1266.81 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | GPCR CalciumGPCRAssays |
| Related | CalciumChannels pHandIonIndicators |
| 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:
 |  | ||||
 | |||||
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 |
αVβ3Integrinregulatesastrocytereactivity
Authors:RaúlLagos-Cabré,AlvaroAlvarez,MileneKong,FrancescaBurgos-Bravo,AreliCárdenas,EdgardoRojas-Mancilla,RamónPérez-Nunez,RodrigoHerrera-Molina,FabiolaRojas,PascalSchneider
Journal:JournalofNeuroinflammation(2017):194
Ca2+signalsinitiateatimmobileIP3receptorsadjacenttoER-plasmamembranejunctions
Authors:NagendraBabuThillaiappan,AlapPChavda,StephenCTovey,DavidLProle,ColinWTaylor
Journal:NatureCommunications(2017):1505
Invivodeeptwo-photonimagingofneuralcircuitswiththefluorescentCa2+indicatorCal-590
Authors:CarstenHTischbirek,AntjeBirkner,ArthurKonnerth
Journal:TheJournalofPhysiology(2016)
Deeptwo-photonbrainimagingwithared-shiftedfluorometricCa2+indicator
Authors:CarstenTischbirek,AntjeBirkner,HongboJia,BertSakmann,ArthurKonnerth
Journal:ProceedingsoftheNationalAcademyofSciences(2015):11377--11382
AAT Bioquest常用产品报价单
| 产品名 | 货号 | 公司分类 | 商城分类 | 美金价 |
| AAT Bioquest/Fluorescein, disodium salt *CAS 518-47-8*/2/100 mg | 2 | iFluor Rapid Tests | 酸碱缓冲液 | 1088.00 | AAT Bioquest/14-3-3 ζ (Ab-58) Antibody/8B0001/50 ug | 8B0001 | Signaling Intermediate Ab | 重组抗体 | 2828.00 | AAT Bioquest/Opioid Receptor (Phospho-Ser375) Antibody/8A0022/50 ug | 8A0022 | Phospho-Specific Ab | 受体 | 2828.00 | AAT Bioquest/iFluor™ 350 goat anti-mouse IgG (H+L)/16440/200 ug | 16440 | Fluorescent Anti-IgGs | 荧光染料 | 1088.00 | AAT Bioquest/trFluor™ Eu goat anti-mouse IgG (H+L)/16518/100 ug | 16518 | Fluorescent Anti-IgGs | 抗小鼠 | 4278.00 | AAT Bioquest/RPE-streptavidin conjugate/16900/100 ug | 16900 | Streptavidin Conjugates | 其他生物染料 | 1378.00 | AAT Bioquest/GCNT3 Antibody/8C14708/50 ug | 8C14708 | Signaling Intermediate Ab | 功能性抗体 | 2828.00 | AAT Bioquest/Amplite™ Luciferase Reporter Gene Assay Kit *Bright Glow*/12518/1 plate | 12518 | Reporter Gene Enzymes | 其它检测试剂盒 | 2103.00 | AAT Bioquest/ReadiLink™ Rapid mFluor™ Violet 450 Antibody Labeling Kit *Microscale Optimized for Labeling 50 &mi | 1100 | mFluor™ Dyes and Kits | 标记试剂盒 | 2103.00 | AAT Bioquest/Amplite™ Colorimetric Urea Quantitation Kit *Blue Color*/10058/200 Tests | 10058 | Diagnostic Molecules | 定量试剂盒 | 2828.00 | AAT Bioquest/Screen Quest™ CHO-Gqi Chimera Cell line/38101/Each | 38101 | cAMP GPCR Assays | 常规细胞株 | 0.00 |
本网站将在规定时间内给予删除等相关处理。
