| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 494/517 |
| MW | 828.54 |
| CAS# | N/A |
| Solvent | Water |
| Storage | F/D/L |
| Category | GPCR CalciumGPCRAssays |
| Related | CalciumChannels pHandIonIndicators BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
UseofFluo-8®AMEsters
1.LoadCellswithFluo-8®AMEsters:
AMestersarethenon-polarestersthatreadilycrosslivecellmembranes,andrapidlyhydrolyzedbycellularesterasesinsidelivecells.AMestersarewidelyusedforloadingavarietyofpolarfluorescentprobesintolivecellnon-invasively.However,cautionsmustbeexcisedwhenAMestersareusedsincetheyaresusceptibletohydrolysis,particularlyinsolution.Theyshouldbereconstitutedjustbeforeuseinhigh-quality,anhydrousdimethylsulfoxide(DMSO).DMSOstocksolutionsmaybestoreddesiccatedat–20°Candprotectedfromlight.Undertheseconditions,AMestersshouldbestableforseveralmonths.
FollowingisourrecommendedprotocolforloadingFluo-8®AMestersintolivecells.Thisprotocolonlyprovidesaguideline,andshouldbemodifiedaccordingtoyourspecificneeds.
a) Preparea2to5mMstocksolutionofFluo-8®AMestersinhigh-quality,anhydrousDMSO.
b) Onthedayoftheexperiment,eitherdissolveFluo-8®inDMSOorthawanaliquotoftheindicatorstocksolutiontoroomtemperature.Prepareaworkingsolutionof1to10µMinHanksandHepesbuffer(HHBS)orthebufferofyourchoicewith0.02%Pluronic®F-127.Formostofcelllines,Fluo-8®reagentswithaconcentrationrangingfrom4-5uMarerecommended.Theexactconcentrationoftheindicatorrequiredforcellloadingmustbedeterminedempirically.Toavoidanyartifactscausedbyoverloadingandpotentialdyetoxicity,itisrecommendedtousetheminimaldyeconcentrationthatcangeneratesufficientsignalstrength.
Note:ThenonionicdetergentPluronic®F-127issometimesusedtoincreasetheaqueoussolubilityofFluo-8®AMesters. AvarietyofPluronic®F-127solutionscanbepurchasedfromAATBioquest.
c) Ifyourcellscontainingtheorganicanion-transports,probenecid(1–2.5mM)orsulfinpyrazone(0.1–0.25mM)maybeaddedtothecellmediumtoreduceleakageofthede-esterifiedindicators.
Note:AvarietyofReadiUse™probenecidincludingwatersolublesodiumsaltandstABIlizedsolutioncanbepurchasedfromAATBioquest.
d) Addequalvolumeofthedyeworkingsolution(fromStepborc)intoyourcellplate.
e) Incubatethedye-loadingplateatacellincubatororroomtemperaturefor20minutestoonehour.
Note:Decreasingtheloadingtemperaturemightreducethecompartmentalizationoftheindictor.
f) ReplacethedyeworkingsolutionwithHHBSorbufferofyourchoice(containingananiontransporterinhibitor,suchas2.5mMprobenecid,ifapplicable)toremoveexcessprobes.
g) RuntheexperimentsatEx/Em =490/525nm
UseofScreenQuest™Fluo-8NWCalciumAssayKitsforHTSApplications
GPCRactivationcanbedetectedbydirectmeasurementofthereceptormediatedcAMPaccumulation,orchangesinintracellularCa2+concentration.GPCRtargetsthatcoupleviaGqproduceanincreaseinintracellularCa2+thatcanbemeasuredusingacombinationofFluo-8®reagentsandafluorescencemicroplatereader.Thefluorescenceimagingplatereaders(suchas,FLIPR™,FDSSorBMGNovoStar™)haveacooledCCDcameraimagingsystemwhichcollectsthesignalfromeachwellofamicroplate(both96and384-well)simultaneously.Theseplatereaderscanreadatsub-secondintervals,whichenablesthekineticsoftheresponsetobecaptured,andhasanintegratedpipettorthatmaybeprogrammedforsuccessiveliquidadditions.BesidestheirrobustapplicationsforGPCRtargets,ourScreenQuest™Fluo-8CalciumAssayKitscanbealsousedforcharacterizingcalciumionchannelsandscreeningcalciumionchannel-targetedcompounds.
Figure2.CarbacholDoseResponsewasmeasuredinHEK-293cellswithScreenQuest™Fluo-8NWAssaykitandFluo-4NWAssayKit.HEK-293cellswereseededovernightat40,000cells/100µL/wellina96-wellblackwall/clearbottomcostarplate.Thegrowthmediumwasremoved,andthecellswereincubatedwith,respectively,100µLoftheScreenQuest™Fluo8-NWcalciumassaykitandFluo-4NWkit(accordingtothemanufacturer’sinstructions)for1houratroomtemperature.Carbachol(25µL/well)wasaddedbyNOVOstar(BMGLabTech)toachievethefinalindicatedconcentrations.TheEC50ofFluo-8NWisabout1.2uM.
ComparedtoothercommercialcalciumassaykitsthateitherbasedonFluo-3orFluo-4,ourScreenQuest™CalciumAssayKitshavethefollowingadvantagesforHTSapplications:
•BroadApplications:workwithbothGPCRandcalciumchanneltargets.
•ConvenientSpectralWavelengths:maximumexcitation@~490nm;maximumemission@~514nm.
•FlexibleDyeLoading:dyeloadingatroomtemperature(ratherthan37ºCrequiredforFluo-4AM).
•NoWashRequiredandNoQuencherInterferencewithYourTargets.
•RobustPerformance:enablecalciumassaysthatareimpossiblewithFluo-4AMorFluo-3AM.
•StrongestSignalIntensity:2timesbrighterthanthatofFluo-4AM;4timesbrighterthanthatofFluo-3AM.
UseofFluo-8®Salts
Calciumcalibrationcanbecarriedoutbymeasuringthefluorescenceintensityofthesaltform(25to50µMinfluorescencemicroplatereaders)oftheindicatorsinsolutionswithpreciselyknownfreeCa2+concentrations.Calibrationsolutionscanbeusedbasedon30mMMOPSEGTACa2+buffer.Ingeneral,watercontainstraceamountofcalciumion.Itishighlyrecommendedtouse30mMMOPS+100mMKCl,pH7.2asbuffersystem.Onecansimplymakea0and39µMcalciumstocksolutionsaslistedbelow,andthese2solutionsareusedtomakeaserialsolutionofdifferentCa2+concentrations
A.0µMcalcium:30mMMOPS+100mMKCl,pH7.2buffer+10mMEGTA
B.39µMcalcium:30mMMOPS+100mMKCl,pH7.2buffer+10mMEGTA+10mMCaCl2
TodetermineeitherthefreecalciumconcentrationofasolutionortheKdofasingle-wavelengthcalciumindicator,thefollowingequationisused:
[Ca]free=Kd[F─Fmin]/Fmax─F]
WhereFisthefluorescenceintensityoftheindicatorataspecificexperimentalcalciumlevel,FministhefluorescenceintensityintheabsenceofcalciumandFmaxisthefluorescenceintensityofthecalcium-saturatedprobe.
Thedissociationconstant(Kd)isameasureoftheaffinityoftheprobeforcalcium.Thecalcium-bindingandspectroscopicpropertiesoffluorescentindicatorsvaryquitesignificantlyincellularenvironmentscomparedtocalibrationsolutions.InsituresponsecalibrationsofintracellularindicatorstypicallyyieldKdvaluessignificantlyhigherthaninvitrodeterminations.InsitucalibrationsareperformedbyexposingloadedcellstocontrolledCa2+buffersinthepresenceofionophoressuchasA-23187,4-bromoA-23187andionomycin.Alternatively,cellpermeabilizationagentssuchasdigitoninorTriton®X-100canbeusedtoexposetheindicatortothecontrolledCa2+levelsoftheextracellularmedium.TheKdvaluesofFluo-8®reagentsarelistedinTable1foryourreference.
| References&Citations |  CitationExplorer |
Below,youmayfindasmallsamplingofspecificFluo-8®AMapplicationssortedbyfieldofstudy.ToinquireaboutapotentialapplicationofFluo-8®AM,ortoconsultwithourfluorescentdyespecialists,pleasecontactusatsupport@aatbio.comor1-800-990-8053.
InOncology,Fluo-8®AMhasbeenusedtostudy:
»BreastcancercellsbymonitoringintracellularCa2+fluxassociatedwithapoptosisandinhibitionby2-aminoethoxydiphenylborate[1]
»Antitumoractivitybywayofthioredoxin-bindingprotein2anditsdependenceonintracellularcalciumconcentration[2]
»Bcl-1andBcl-2regulationthroughcharacterizationofcytosolictransportasquantifiedbycalciumflux[3]
»Ca2+influxandCa2+channelactivityinNCI-H460cellsasaparameterformonitoringprogressionofnon-smallcelllungcancer[4]
»Ca2+releasebyHN4cellsandCLIC4upregulationofapoptosisthroughmitochondrialandendoplasmicreticulumpathways[5]
InCardiology,Fluo-8®AMhasbeenusedtostudy:
»Low-energyfar-fieldstimulationasatherapyfortachycardiaandfibrillation[6]
»Calciumfluxduringcalciumsparksinventricularmyocytes[7]
»Cardiacconductionasafunctionofcellrigidityinthecontextofcardiovasculardisease[8]
»DiastolicCa2+transientsincardiacmyocytesandSR-luminalandfreecytoplasmicCa2+concentrations[9]
»Sphingosine-1-phosphate(S1P)receptoractivationinvalvularinterstitialcellsasdetectedbycytosolicCa2+flux[10]
InNeurobiology,Fluo-8®AMhasbeenusedtostudy:
»HippocampalCA1neurons,visualizatingneuronstoinvestigatetheroleofamyloid-βintheprogressionofAlzheimer"sdisease[11]
»CytosolicCa2+concentrationsinHEK293cellsanditsregulatoryeffectonAβ1-42andhAmylinandassociatedsignalingpathways[12]
»Gprotein-coupledreceptors(GPRs)inresponsetocannabinoidsinpresynapticCA3orpostsynapticCA1pyramidalcells[13]
»Medullaryinterneuronsanddendriticcalciumactivityinregardstoinspiratorybursts[14]
»N2acellactivationbyhistamine,asmonitoredbyincreasesinintracellularCa2+concentrations[15]
InStemCells,Development&Differentiation,Fluo-8®AMhasbeenusedtostudy:
»Inductionofpluripotentstemcells(iPSCs)intofunctioningcardiaccells,asvalidatedbyCa2+fluxandmembranepotential[16]
»CXCR4andCXCR7receptorsinTcellsandtheirroleincellsurvivalandchemotaxis[17]
»Ca2+uptakebymyocytesderivedfromhumaninducedpluripotentstemcellsduringpathogenesisofDuchennemusculardystrophy[18]
»AgoNIST-inducedcalciumtransientsindifferentiationofratbonemarrowmesenchymalstemcellsintosmoothmusclecells[19]
»CalciumchannelblockadesandtheireffectoncardiacProgenitorcellproliferationanddifferentiation[20]
AAT Bioquest AAT Bioquest是一家位于美国的生物公司，前身为ABD Bioquest，总部位于加利福尼亚州。专门从事光学检测技术十多年，一直致力于光谱学检测领域技术的创新和突破。其独特的光学检测技术，综合了化学、生物学和信息学等各个领域的研究，引领了比色、荧光和发光技术新一代光学探针的浪潮。AAT Bioquest在全球拥有强大的经验丰富的专业分销商网络，为从小型研究机构到《财富》500强企业的各类客户提供卓越的产品和定制服务。
美国AATBioquestInc.(前身是ABDBioquest，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。AATBioquest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。AATBioquest会不断介绍新产品，快速的丰富各个领域的产品。
1）我们提供反应荧光探针和发光探针，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）我们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）我们不断的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）我们致力于开发用于信号转导研究的试剂；5）我们提供生理和神经探针，特别是钙离子指示剂和膜电位探针。
作为AATBioquestInc.的中国区域代理，艾美捷科技为中国客户提供光谱学检测领域，包括吸收（颜色），荧光和发光技术等全系列解决方案。我们也将一如既往更加努力为国内用户提供快捷、方便的高质量产品，同时更为您售前售后全面技术支持。
AATBioquest,Inc.(formerlyABDBioquest,Inc.)develops,manufacturesandmarketsbioanalyticalresearchreagentsandkitstoscientistsengagedinlifesciencesresearch,diagnosticR&Danddrugdiscovery.Wespecializeintheareaofphotometricdetectionsincludingabsorption(color),fluorescenceandluminescencetechnologies.TheCompany"sproductsenablescientistsandbiomedicalresearcherstobetterunderstandbiochemistry,immunology,cellBIOLOGyandmolecularbiology.AATBioquestconstantlyintroducesnewproducts,andoffersarapidlyexpandinglistofproductsthataregroupedintoseveralproductlines.
1)Ourreactivefluorescentandluminescentprobes,biotinsandtagenzymesareusedforlabelingsmalldrugmoleculesandbiopolymers,e.g,proteins,nucleicacidsandcarbohydrates;2)Wedevelopfluorescentandluminescentprobesfordetectingproteins,nucleicacidsandlivecells;3)Weconstantlyintroducenovelfluorescentandluminescentprobesfordetectingvariousenzymes,inparticular,hydrolyticandredoxenzymes;4)Wefocusondevelopingreagentsforsignaltransductionresearch;and5)Wealsoofferphysiologicalandneurologicalprobes,e.g.,calciumindicatorsandmembranepotentialprobes.
Besidesthestandardcatalogproductswealsooffercustomservicetomeetyourspecialresearchneeds.Ourcurrentservicesincludecustomsynthesisofcolorimetric,fluorescentandluminescentprobes,customdevelopmentofbiochemical,cell-basedanddiagnosticassaysandcustomscreeningofyourcompoundlibrariesagainstyourdefinedtargetsusingourvalidatedHTSassays.
ebiomall.com
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请各位大侠给予帮助！！
谢谢！！
而且样品中的无水硫酸钠未变色,而做标准曲线的五个和空白对照的变为蓝色了,请高手指教,多谢!
还有,是否变蓝对测定结果有影响吗?
谢了哈
欢迎你！请下次规范发贴:)
二苯胺
甲基绿派洛宁
还有一种忘了。
假期没法联系老师呵呵。
这三种的作用都是什么。
健那绿染液是一种活体染液，实验对象必须是活细胞，健那绿可以使活细胞中的线粒体呈现蓝绿色，而细胞质接近无色。
1、快讯上是Invotrogen公司的SYRBGOLD，不过500ul/1390元，实在有点贵，为了查用量；
2、结果又查出赛百盛的GoldViewTMDNA染料1ml/100元；
3、还有一种在日光下即可看出DNA条带的上海华舜生物工程有限公司的LightBluedye；
为了一个EB，居然不小心查出三种染料，而对这三种染料的评价也褒贬不一，我个人希望找个毒害小些，而且灵敏度还不能低于EB，价格方面也能让老板接受的来。
而对这三个染料，我有三个问题：
1、SYRBGOLD的用量问题，500ul能用到200次吗（我一般一次用30ml的胶，配在胶中）？
2、GoldViewTMDNA染料的成分是否如有些人所说的就是丫啶橙（能举出确切证据吗）？
3、LightBluedye灵敏度如何？能比得过EB吗？
前一阵子实验室开始使用一种新的核酸染料-goldview,取代原先用的溴化乙锭-EB.goldview现在是由赛百盛出售.以下摘自官方网站:
GoldViewTM核酸染料——使用说明
概述
GoldViewTM是一种可代替溴化乙锭（EB）的新型核酸染料，采用琼脂糖电泳检测DNA时，GoldViewTM与核酸结合后能产生很强的荧光信号，其灵敏度与EB相当，使用方法与之完全相同。在紫外透射光下双链DNA呈现绿色荧光，而单链DNA呈红色荧光。GoldView不仅能染DNA，也可用于染RNA。
通过Ames试验、小鼠骨髓嗜多染红细胞微核试验、小鼠睾丸精母细胞染色体畸变试验，致突变性结果均为阴性；而溴化乙锭（EB）是一种强致癌剂。因此用GoldviewTM代替EB不失为一种明智的选择。
使用方法
1.将100ml琼脂糖凝胶溶液（浓度一般为0.8%~2%）在微波炉中融化。
2.加入5µlGoldView，轻轻摇匀，避免产生气泡。
3.冷却至不烫手时倒胶，待琼脂糖凝胶完全凝固后上样电泳。
4.电泳完毕在紫外灯下观察。若使用数码相机照像记录，则关闭相机的闪光灯，放在自动档即可；若使用凝胶成像系统照相，通过调节光圈、曝光时间，选择合适的滤光片，可得到成像清晰、背景较低的照片。
注意事项
1.胶厚度不宜超过0.5cm，胶太厚会影响检测的灵敏度。
2.加入GoldView的琼脂糖凝胶反复融化可能会对核酸检测的灵敏度产生一定影响，但不明显。
3.通过凝胶电泳回收DNA片段时，建议使用GoldView染色，在自然光下切割DNA条带，避免紫外线与EB对目的DNA产生的损伤，可明显提高克隆、转化、转录等分子生物学下游操作的效率。
4.虽然未发现GoldView有致癌作用，但对皮肤、眼睛会有一定的刺激，操作时应戴上手套。
电泳结果显示GV灵敏度与EB相当
问题1.Goldview到底是不是丫啶橙？
2.赛百盛不公布其成分的原因是害怕其为丫啶橙还是出于技术保密？
健那绿——高中唯一一个活体染色剂。染线粒体的，染成蓝绿色
2、苏丹三 脂肪 橙红
3、苏丹四 脂肪 红
4、双缩脲 蛋白质 紫
5、龙胆紫 染色质 紫
6、碘 淀粉 蓝
7、健那绿 线粒体 绿
8、甲基绿 DNA 绿
9、吡罗红 RNA 红
10、溴麝香草酚蓝 CO2 由蓝变绿再变黄
11、重铬酸钾 酒精 酸性条件下由橙色变成灰绿
12、醋酸洋红（龙胆紫、改良苯酚品红） 染色质 红
13、台盼蓝 检验活死细胞 死细胞会被染成蓝色（不常用）
