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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/Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit/36318/10 Plates
产品编号:36318
市  场 价:¥13900.00
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:$695.00
品      牌: AAT Bioquest
公      司:AAT Bioquest
公司分类:
AAT Bioquest/Screen Quest™ Calbryte-520 Probenecid-Free and Wash-Free Calcium Assay Kit/36318/10 Plates
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
CalciumfluxassaysarethepreferredmethodsindrugdiscoveryforscreeningGproteincoupledreceptors(GPCR).ScreenQuest™Calbryte-520Probenecid-FreeandWash-FreeCalciumAssayKitprovidesthemostrobusthomogeneousfluorescence-basedassayfordetectingtheintracellularcalciummobilization.CellsexpressingaGPCRofinterestthatsignalsthroughcalciumarepre-loadedwithourproprietaryCalbryte™-520NWwhichcancrosscellmembrane.Calbryte™-520NWisthebrightestcalciumindicatoravailableforHTSscreening.Onceinsidethecell,thelipophilicblockinggroupsofCalbryte™-520NWarecleavedbynon-specificcellesterase,resultinginanegativelychargedfluorescentdyethatstaysinsidecells,anditsfluorescenceisgreatlyenhanceduponbindingtocalcium.Whencellsstimulatedwithscreeningcompounds,thereceptorsignalsreleaseofintracellularcalcium,whichgreatlyincreasethefluorescenceofCalbryte™-520NW.Thecharacteristicsofitsexcellentcellretention,highsensitivity,and100-250timesfluorescenceincreases(whenitformscomplexeswithcalcium)makeCalbryte™-520NWanidealindicatorformeasurementofcellularcalcium.Calbryte™-520NWistheonlycalciumdyethatdoesnotrequireprobenecidforbettercellularretention.ThisScreenQuest™Calbryte-520Probenecid-FreeandWash-FreeCalciumAssayKitprovidesthemostoptimizedassaymethodformonitoringG-protein-coupledreceptors(GPCRs)andcalciumchannelswithfragileordifficultcelllines.Theassaycanbeperformedinaconvenient96-wellor384-wellmicrotiter-plateformatandeasilyadaptedtoautomation.
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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. PrepareCalbryte™520NWdye-loADIngsolution:
    1. Thawallthekitcomponentsatroomtemperaturebeforeuse.
    2. MakeCalbryte™520NWstocksolution:Add20µL(forCat.#36317)or200µL(forCat.#36318and#36319)ofDMSOintothevialofCalbryte™520NW(ComponentA),andmixthemwell.
      Note:20µLofCalbryte™520NWstocksolutionisenoughforoneplate.UnusedCalbryte™520NWstocksolutioncanbealiquotedandstoredat<-20=""oc=""for=""more=""than=""one=""month=""if=""the=""tubes=""are=""sealed=""tightly.=""protect=""from=""light=""and=""avoid=""repeated=""freeze-thaw="">
    3. Make1Xassaybuffer:Mix9mLofHHBS(ComponentC,notincludedinthekitcat#36319)with1mLof10XPluronic®F127Plus(ComponentB),andmixthemwell.
    4. MakeCalbryte™520NWdye-loadingsolutionforonecellplate:Add20µLofCalbryte™520NWstocksolution(fromStep2.2)into10mLof1Xassaybuffer(fromStep2.3),andmixthemwell.Thisworkingsolutionisstableforatleast2hoursatroomtemperature.
  3. Runcalciumassay:
    1. Add100µL/well(96-wellplate)or25µL/well(384-wellplate)ofCalbryte™520NWdye-loadingsolution(fromStep2.4)intothecellplate.
    2. Incubatethedye-loadingplateinacellincubatorfor30minutes,andthenincubatetheplateatroomtemperatureforanother15-30minutes.
      Note1:Iftheassayrequires37oC,performtheexperimentimmediatelywithoutfurtherroomtemperatureincubation.
      Note2:Ifthecellscanfunctionwellatroomtemperatureforlongertime,incubatethecellplateatroomtemperaturefor1hour(Itisrecommendedthattheincubationtimebenolongerthan2hours.)
    3. PreparethecompoundplatewithHHBSoryourdesiredbuffer.
    4. RunthecalciumfluxassaybymonitoringthefluorescenceintensityatEx/Em=490/525nm.
References&Citations
CitationExplorer

CalreticulinregulatesTGF-β1-inducedepithelialmesenchymaltransitionthroughmodulatingSmadsignalingandcalciumsignaling
Authors:YanjiaoWu,XiaoliXu,LunkunMa,QianYi,WeichaoSun,LilingTang
Journal:TheInternationalJournalofBiochemistry&CellBIOLOGy(2017)

Dexmedetomidinereduceshypoxia/reoxygenationinjurybyregulatingmitochondrialfissioninrathippocampalneurons
Authors:JiaLiu,QingDu,HeZhu,YuLi,MaodongLiu,ShoushuiYu,ShileiWang
Journal:IntJClinExpMed(2017):6861--6868

Monosialoganglioside1mayalleviateneurotoxicityinducedbypropofolcombinedwithremifentanilinneuralstemcells
Authors:JiangLu,Xue-qinYao,XinLuo,YuWang,SookjaKimChung,He-xinTang,ChiWaiCheung,Xian-yuWang,ChenMeng,QingLi
Journal:NeuralRegenerationResearch(2017):945

Obtainingspontaneouslybeatingcardiomyocyte-likecellsfromadipose-derivedstromalvascularfractionsculturedonenzyme-crosslinkedgelatinhydrogels
Authors:GangYang,ZhenghuaXiao,XiaomeiRen,HaiyanLong,KunlongMa,HongQian,YingqiangGuo
Journal:ScientificReports(2017):41781

Di(2-ethylhexyl)phthalate-inducedapoptosisinratINS-1cellsisdependentonactivationofendoplasmicreticulumstressandsuppressionofantioxidantprotection
Authors:XiaSun,YiLin,QianshengHuang,JunpengShi,LingQiu,MeiKang,YajieChen,ChaoFang,TingYe,SijunDong
Journal:Journalofcellularandmolecularmedicine(2015):581--594

Theeffectofmitochondrialcalciumuniporteronmitochondrialfissioninhippocampuscellsischemia/reperfusioninjury
Authors:LantaoZhao,ShuhongLi,ShileiWang,NingYu,JiaLiu
Journal:Biochemicalandbiophysicalresearchcommunications(2015):537--542

FungusinducesthereleaseofIL-8inhumancornealepithelialcells,viaDectin-1-mediatedproteinkinaseCpathways.
Authors:Xu-DongPeng,Gui-QiuZhao,JingLin,NanJiang,QiangXu,Cheng-ChengZhu,Jain-QiuQu,LinCong,HuiLi
Journal:Internationaljournalofophthalmology(2014):441--447

Propofolandremifentanilatmoderateandhighconcentrationsaffectproliferationanddifferentiationofneuralstem/Progenitorcells
Authors:QingLi,JiangLu,XianyuWang
Journal:Neuralregenerationresearch(2014):2002

Roleofmitochondrialcalciumuniporterinregulatingmitochondrialfissioninthecerebralcortexesoflivingrats
Authors:NanLiang,PengWang,ShileiWang,ShuhongLi,YuLi,JinyingWang,MinWang
Journal:JournalofNeuralTransmission(2014):593--600

Increasedexpressionofcelladhesionmolecule1bymastcellsasacauseofenhancednerve--mastcellinteractioninahapten-inducedmousemodelofatopicdermatitis
Authors:MHagiyama,TInoue,TFuruno,TIino,SItami,MNakanishi,HAsada,YHosokawa,AIto
Journal:BritishJournalofDermatology(2013):771--778


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