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主营:研究并生产荧光和发光探针,信号转导研究的试剂
℡ 4000-520-616
℡ 4000-520-616
AAT Bioquest/Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Assay Kit *Optimized for microplate reader*/3631
产品编号:36312
市  场 价:¥114560.00
场      地:美国(厂家直采)
联系QQ:1570468124
电话号码:4000-520-616
邮      箱: info@ebiomall.com
美  元  价:$5728.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Cell Meter™ No Wash and Probenecid-Free Endpoint Calcium Assay Kit *Optimized for microplate reader*/3631
商品介绍
Overview
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Ex/Em(nm)490/525
MWN/A
CAS#N/A
SolventN/A
StorageF/D/L
CategoryGPCR
CalciumGPCRAssays
RelatedCalciumChannels
pHandIonIndicators
CellMeter™NoWashandProbenecid-FreeEndpointCalciumAssayKitenableshomogeneousfluorescence-basedassaysfordetectingintracellularcalciummobilizationwithouttheneedtousekineticsreadingmode.ItcanbeusedonFluorescencemicroplatereaderswithbottomreadmodethatdonothaveabuild-inliquiddispenserortheabilityofkineticsreading.AfterloadingtheFluo-8E™AMdyeintocellsofinterest,withoutwashsteps,onecansimplyaddthecalciumfluxagonistbyaliquiddispenserorhandpipetting,andthenreadtheplatebyafluorescentreaderatEx/Em=490/525nm.Fluo-8E™AMcancrosscellmembranepassivelybydiffusion.Onceinsidethecells,thelipophilicblockinggroupsofFluo-8E™AMarecleavedbyesterase,resultinginanegativelychargedfluorescentdyethatstaysinsidecells.Itsfluorescenceisgreatlyenhancedandlonglastinguponbindingtocalcium.WhencellsexpressingGPCRofinterestarestimulatedwithanagonist,thereceptorsignalsthereleaseofintracellularcalcium,whichsignificantlyincreasesthefluorescenceofFluo-8E™.Thecharacteristicsofitshighsensitivity,>100timesfluorescenceenhancementandlonglastingfluorescentsignalmakeFluo-8E™anidealindicatorforthemeasurementofcellularcalciumonfluorescencemicroplatereadersthatdonothavethefluidtransferandkineticreADIngmodecapABIlity.TheCellMeter™NoWashandProbenecid-FreeEndpointCalciumAssayKitcanbeperformedina96-wellor384-wellmicrotiter-plateformat.
SpectrumAdvancedSpectrumViewer

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.
  1. Preparecells:
    1. Foradherentcells:Platecellsovernightingrowthmediumat40,000to80,000cells/well/100µLfora96-wellplateor10,000to20,000cells/well/25µLfora384-wellplate.
    2. Fornon-adherentcells:CentrifugethecellsfromtheculturemediumandthensUSPendthecellpelletinCellgrowthmediumorHHBSat125,000to250,000cells/well/100µLfora96-wellpoly-Dlysineplateor30,000to60,000cells/well/25µLfora384-wellpoly-Dlysineplate.Centrifugetheplateat800rpmfor2minuteswithbrakeoffpriortotheexperiments.
      Note:Eachcelllineshouldbeevaluatedontheindividualbasistodeterminetheoptimalcelldensityfortheintracellularcalciummobilization

  2. PrepareFluo-8E™AMdye-loadingsolution(foroneplate):
    1. MakeFluo-8E™AMstocksolution:Add10µLofDMSOintoonevialofFluo-8E™AM(ComponentA),andmixthemwell.
      Note:10µLofFluo-8E™AMstocksolutionisenoughfor50assays(halfofthe96-wellplate).UnusedFluo-8E™AMstocksolutioncanbealiquotedandstoredat<-20=""°c=""for=""more=""than=""one=""month=""if=""the=""tubes=""are=""sealed=""tightly.=""protect=""from=""light=""and=""avoid=""repeated=""freeze-thaw="">
    2. Make1Xassaybuffer:Make1Xassaybufferbymixing9mLofHHBS(ComponentC)into10XPluronic®F127Plus(1mL,ComponentB),andmixthemwell.
      Note:10mLof1Xassaybufferisenoughforoneplate.Aliquotandstoreunused1Xassaybufferat<-20=""°c.=""protect=""from=""light=""and=""avoid=""repeated=""freeze-thaw="">
    3. MakeFluo-8E™AMdye-loadingsolutionfor1/2cellplate:Add10µLofFluo-8E™AMDMSOstocksolution(fromStep2.1)into5mLof1Xassaybuffer(fromStep2.3),andmixthemwell.Thisworkingsolutionisstableforatleast2hoursatroomtemperature.

  3. Runcalciumassay:
    1. Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofFluo-8E™AMdye-loadingsolution(fromStep2.4)intothecellplate.Donotremovethegrowthmediumfromthecellplate.
    2. Incubatethedye-loadingplateinacellincubatorfor45-60minutes.
    3. Prepare5XagoNISTcompoundwithHHBSoryourdesiredbuffer.
    4. Add50µLofthepreparedagonistfromstep3.3andrunthecalciumfluxassayimmediatelybymonitoringthefluorescenceintensityatEx/Em=490/525nmwithbottomreadmode.
      Note:Toachievethebestresults,itisimportanttoruntheassaywithin1minuteaftertheadditionoftheagonist.Itisalsoimportanttomakesurethetimebetweentheagonistadditionandthebeginningoftheactualreadingstaysconstantforallthesamples.
References&Citations
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1.   TerritoPR,HeilJ,BoseS,EvansFJ,BalabanRS.(2007)Fluorescenceabsorbanceinner-filterdecomposition:theroleofemissionshapeonestimatesoffreeCa(2+)usingRhod-2.ApplSpectrosc,61,138.

2.   BednarB,CunninghamME,KissL,ChengG,McCauleyJA,LivertonNJ,KoblanKS.(2004)KineticcharacterizationofnovelNR2Bantagonistsusingfluorescencedetectionofcalciumflux.JNeurosciMethods,137,247.

3.   MartinVV,BeierleinM,MorganJL,RotheA,GeeKR.(2004)Novelfluo-4analogsforfluorescentcalciummeasurements.CellCalcium,36,509.

4.   StammC,delNidoPJ.(2004)ProteinkinaseCandmyocardialcalciumhandlingduringischemiaandreperfusion:lessonslearnedusingRhod-2spectrofluorometry.ThoracCardiovascSurg,52,127.

5.   StammC,FriehsI,ChoiYH,ZurakowskiD,McGowanFX,delNidoPJ.(2003)Cytosoliccalciumintheischemicrabbitheart:assessmentbypH-andtemperature-adjustedrhod-2spectrofluorometry.CardiovascRes,59,695.

6.   DuC,MacGowanGA,FarkasDL,KoretskyAP.(2001)CalibrationofthecalciumdissociationconstantofRhod(2)intheperfusedmouseheartusingmanganesequenching.CellCalcium,29,217.

7.   DuC,MacGowanGA,FarkasDL,KoretskyAP.(2001)Calciummeasurementsinperfusedmouseheart:quantitatingfluorescenceandabsorbanceofRhod-2byapplicationofphotonmigrationtheory.BiophysJ,80,549.

8.   LannergrenJ,WesterbladH,BrutonJD.(2001)ChangesinmitochondrialCa2+detectedwithRhod-2insinglefrogandmouseskeletalmusclefibresduringandafterrepeatedtetaniccontractions.JMuscleResCellMotil,22,265.

9.   MacGowanGA,DuC,GlontyV,SuhanJP,KoretskyAP,FarkasDL.(2001)Rhod-2basedmeasurementsofintracellularcalciumintheperfusedmouseheart:cellularandsubcellularlocalizationandresponsetopositiveinotropy.JBiomedOpt,6,23.

10.   MurielMP,LambengN,DariosF,MichelPP,HirschEC,AgidY,RubergM.(2000)Mitochondrialfreecalciumlevels(Rhod-2fluorescence)andultrastructuralalterationsinneuronallydifferentiatedPC12cellsduringceramide-dependentcelldeath.JCompNeurol,426,297.


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