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当前位置: 首页 > 产品中心 > Other_biological_dyes > AAT Bioquest/Fluo-5F,五钾盐*细胞防渗剂*/20562/500微克
商品详细AAT Bioquest/Fluo-5F,五钾盐*细胞防渗剂*/20562/500微克
AAT Bioquest/Fluo-5F,五钾盐*细胞防渗剂*/20562/500微克
AAT Bioquest/Fluo-5F,五钾盐*细胞防渗剂*/20562/500微克
商品编号: 20562
品牌: aatbio
市场价: ¥56560.00
美元价: 33936.00
产地: 美国(厂家直采)
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产品分类: 其他生物染料
公司分类: Other_biological_dyes
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Ex/Em(nm)494/516
MW931.05
CAS#N/A
SolventWater
Storage
CategoryGPCR
CalciumGPCRAssays
RelatedCalciumChannels
pHandIonIndicators
Fluo-5FisananalogofFluo-4withlowercalcium-bindingaffinity(Kd=~2.3uM),makingitsuitablefordetectingintracellularcalciumlevelsintherangeof1µMto1mMthatwouldsaturatetheresponseofFluo-4.Cellsmaybephysicallyloadedwiththecell-impermeantFluo-5Fusingpatchpipetteormicroinjection.MorecommonlyFluo-5FAMester(#20560)maybedirectlyloadedintolivecellsbyaddingthedissolvedindicatordirectlytodishescontainingtheculturedcells.Itiscompatiblewithexcitationat488nmbyargon-ionlasersources,makingFluo-5Fusefulforconfocalmicroscopy,flowcytometry,andmicroplatescreeningapplications.Ithasexcitationandemissionwavelengthsat494and516nmrespectively.Uponcalciumbinding,itsfluorescenceintensityincreasesby>100fold.
SpectrumAdvancedSpectrumViewer

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Movemouseovergridtodisplaywavelength&intensityvalues.

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Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

UseofCalciumindicatorAMEsters

 

1.LoadCellswithCalciumIndicatorAMEsters:

AMestersarethenon-polarestersthatreADIlycrosslivecellmembranes,andrapidlyhydrolyzedbycellularesterasesinsidelivecells.AMestersarewidelyusedforloadingavarietyofpolarfluorescentprobesintolivecellnon-invasively.However,cautionsmustbeexcisedwhenAMestersareusedsincetheyaresusceptibletohydrolysis,particularlyinsolution.Theyshouldbereconstitutedinhigh-quality,anhydrousdimethylsulfoxide(DMSO).DMSOstocksolutionsshouldbestoreddesiccatedat-20°Candprotectedfromlight.Undertheseconditions,AMestersshouldbestableforseveralmonths.

 

FollowingisourrecommendedprotocolforloadingAMestersintolivecells.Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.

a)      Preparea2to5mMAMestersstocksolutioninhigh-quality,anhydrousDMSO.

b)      Onthedayoftheexperiment,eitherdissolvecalciumindicatorssolidinDMSOorthawanaliquotoftheindicatorstocksolutionstoroomtemperature.Prepareaworkingsolutionof2to20µMinthebufferofyourchoice(suchasHanksandHepesbuffer)with0.04%Pluronic®F-127.Formostcelllineswerecommendthefinalconcentrationofcalciumindicatorsbe4-5uM.Theexactconcentrationofindicatorsrequiredforcellloadingmustbedeterminedempirically.Toavoidanyartifactscausedbyoverloadingandpotentialdyetoxicity,itisrecommendedtousetheminimalprobeconcentrationthatcanyieldsufficientsignalstrength.

Note:ThenonionicdetergentPluronic®F-127issometimesusedtoincreasetheaqueoussolubilityofcalciumindicatorAMesters. AvarietyofPluronic®F-127solutionscanbepurchasedfromAATBioquest.

c)      Ifyourcells(suchasCHOcells)containingtheorganicanion-transports,probenecid(2–5mM)orsulfinpyrazone(0.2–0.5mM)maybeaddedtothethedyeworkingsolution(finalinwellconcentrationwillbe1-2.5mMforprobenecid,or0.1-0.25mMforsulfinpyrazone)toreducetheleakageofthede-esterifiedindicators.

Note:AvarietyofReadiUse™probenecidincludingwatersolublesodiumsaltandstABIlizedsolutioncanbepurchasedfromAATBioquest

d)      Addequalvolumeofthedyeworkingsolution(fromStepborc)intoyourcellplate.

e)      Incubatethedye-loadingplateroomattemperatureor37°Cfor20minutes(especiallyFluo-8AM)to2hours,andthenincubatetheplateatroomtemperatureforanother30minutes.

Note1:Decreasingtheloadingtemperaturemightreducethecompartmentalizationoftheindictor.

Note2:IncubatetheCal-520AMlongerthan2hoursgivesbettersignalintensityforsomecelllines.

f)       ReplacethedyeworkingsolutionwithHHBSorbufferofyourchoice(containingananiontransporterinhibitor,suchas1mMprobenecid,ifapplicable)toremoveexcessprobes.

g)      RuntheexperimentsatdesiredEx/Emwavelengths(seeTable1).

 

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.

 

UseofCalciumindicatorSalts

TodetermineeitherthefreecalciumconcentrationofasolutionortheKdofasingle-wavelengthcalciumindicator,thefollowingequationisused:

[Ca]free=Kd[F-Fmin]/Fmax-F]

WhereFisthefluorescenceoftheindicatoratexperimentalcalciumlevels,FministhefluorescenceintheabsenceofcalciumandFmaxisthefluorescenceofthecalcium-saturatedprobe.Thedissociationconstant(Kd)isameasureoftheaffinityoftheprobeforcalcium.TheCa2+-bindingandspectroscopicpropertiesoffluorescentindicatorsvaryquitesignificantlyincellularenvironmentscomparedtocalibrationsolutions.InsitucalibrationsofintracellularindicatorstypicallyyieldKdvaluessignificantlyhigherthaninvitrodeterminations.InsitucalibrationsareperformedbyexposingloadedcellstocontrolledCa2+buffersinthepresenceofionophoressuchasA-23187,4-bromoA-23187andionomycin.Alternatively,cellpermeabilizationagentssuchasdigitoninorTriton®X-100canbeusedtoexposetheindicatortothecontrolledCa2+levelsoftheextracellularmedium.TheKdvaluesofsomecalciumreagentsarelistedinTable1foryourreference.

 

UseofCalciumindicatorConjugates

 

Comparedtothefreeionindicator,dextranconjugatesofthesesameindicatorsexhibitbothreducedcompartmentalizationandmuchlowerratesofdyeleakage.Sincethemolecularweightofthedextran,netcharge,degreeoflabeling,andnatureofthedyemayaffecttheexperiment,researchersareadvisedtoconsulttheprimaryliteratureforinformationspecifictotheapplicationofinterest.

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


品牌介绍
美国AAT Bioquest Inc.(前身是ABD Bioquest,Inc.)是一家为从事生命科学研究、诊断研发及药物开发的科学家研发、生产和销售生物分析研究试剂和试剂盒的公司。公司致力于光谱学检测领域,包括吸收(颜色)、荧光和发光技术。AAT Bioquest的产品帮助全世界的科学家和生物医药研究者更好的了解生物化学、免疫学、细胞生物学和分子生物学等领域。AAT Bioquest会不断研发新产品,快速地丰富各个领域的产品。 1)提供反应探针和发光探针,生物素和端粒酶能够应用于标记药物小分子和生物聚合物,如蛋白、核酸以及其他碳水化合物; 2)研究并生产荧光和发光探针用于检测蛋白,核酸和活细胞; 3)不断推出新型的荧光和发光探针用于检测多种酶,特别是检测水解酶和氧化还原酶类; 4)致力于开发用于信号转导研究的试剂; 5)提供生理和神经探针,特别是钙离子指示剂和膜电位探针。
品牌分类
淋巴细胞信号传导 GPCR抗体 羰基活性生物素 细胞代谢 胺反应生物素 链霉亲和素结合物 生物素化抗IgGs 乙酰基特异性抗体 细胞骨架抗体
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