| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 524/551 |
| MW | 1015.96 |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | GPCR CalciumGPCRAssays |
| Related | CalciumChannels pHandIonIndicators BiochemicalAssays |
| Spectrum | AdvancedSpectrumViewer |
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 |
Effectofstemcellnicheelasticity/ECMproteinontheself-beatingcardiomyocytedifferentiationofinducedpluripotentstem(iPS)cellsatdifferentstages
Authors:MitsuhiHirata,TetsujiYamaoka
Journal:ActaBiomaterialia(2017)
Emerinplaysacrucialroleinnuclearinvaginationandinthenuclearcalciumtransient
Authors:MasayaShimojima,ShinsukeYuasa,ChikaakiMotoda,GakutoYozu,ToshihiroNagai,ShogoIto,MarkLachmann,ShinKashimura,MakotoTakei,DaiKusumoto
Journal:ScientificReports(2017)
Preliminaryfindingsonultrasoundmodulationoftheelectromechanicalfunctionofhumanstem-cell-derivedcardiomyocytes
Authors:AndrewWilliamChen,AleksandraKlimas,VesnaZderic,IvanSuaresCastellanos,EmiliaEntcheva
Journal:(2017):1--4
TheroleofspatialorganizationofCa(2+)releasesitesinthegenerationofarrhythmogenicdiastolicCa(2+)releaseinmyocytesfromfailinghearts.
Authors:AndriyEBelevych,Hsiang-TingHo,IngridMBonilla,RadmilaTerentyeva,KarstenESchober,DmitryTerentyev,CynthiaACarnes,SándorGyörke
Journal:Basicresearchincardiology(2017):44
Dynamicpolyrotaxane-coatedsurfaceforeffectivedifferentiationofmouseinducedpluripotentstemcellsintocardiomyocytes
Authors:Ji-HunSeo,MitsuhiHirata,SachiroKakinoki,TetsujiYamaoka,NobuhikoYui
Journal:RSCAdvances(2016):35668--35676
IndividualevaluationofcardiacMarkerexpressionandself-beatingduringcardiacdifferentiationofP19CL6cellsondifferentculturesubstrates
Authors:TetsujiYamaoka,MitsuhiHirata,TakaakiDan,AtsushiYamashita,AkihisaOtaka,TakahikoNakaoki,AziziMiskon,SachiroKakinoki,AtsushiMahara
Journal:JournalofBiomedicalMaterialsResearchPartA(2016)
Involvementofaberrantcalciumsignallinginherpeticneuralgia
Authors:RebekahAWarwick,MenachemHanani
Journal:Experimentalneurology(2016):10--18
MultiplepathwaysforelevatingextracellularadenosineintherathippocampalCA1regioncharacterizedbyadenosinesensorcells
Authors:KunihikoYamashiro,YukiFujii,ShoheiMaekawa,MitsuhiroMorita
Journal:JournalofNeuRochemistry(2016)
OptoDyCEasanautomatedsystemforhigh-throughputall-opticaldynamiccardiacelectrophysiology
Authors:AleksandraKlimas,ChristinaMAmbrosi,JinzhuYu,JohnCWilliams,HaroldBien,EmiliaEntcheva
Journal:Naturecommunications(2016)
TheGprotein-coupledreceptorGPR157regulatesneuronaldifferentiationofradialglialProgenitorsthroughtheGq-IP3pathway
Authors:YutakaTakeo,NobuhiroKurabayashi,MinhDangNguyen,KamonSanada
Journal:Scientificreports(2016)
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！！
取片剂一片，照溶出度测定法（中国药典2000年版二部附录ⅩC第三法），以水250ml为溶剂，转速为每分钟50转，依法操作，分别经15、30、45、60、75、210分钟取溶液滤过，精密量取续滤液5ml于分液漏斗中，加入pH7.4磷酸盐缓冲液5ml，5ml0.3%溴麝香草酚蓝溶液，用15ml氯仿分两次萃取，合并萃取液，加入0.4g无水硫酸钠，照分光光度法（中国药典2000年版二部附录ⅥA），在410nm波长处测得溶出A值。现15、30、45、60、75、210分钟溶出A值分别为0.0413、0.0544、0.0437、0.0479、0.0394、0.0302。（测定吸收度偏小是否不准，有影响。）
请各位站友指教。
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。
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:)
产品主要应用：点击化学(Clickchemistry)、蛋白质组学研究中的双向荧光差异凝胶电泳(2DDIGE)和实时荧光定量PCR(RealtimePCR)。
氨基类染料是包含自由氨基的活性染料，染料可与活化羧酸衍生物和其他亲电子的试剂结合。比如：氨基与EDC-活化的羧基结合。
相关产品如下：
中文名英文名产品编号分子结构Cy7.5胺Cy7.5amineAGF1350A[img]/KindEditor_4.0.1/attached/image/20130704/20130704110804_5250.jpg[/img]Cy5胺Cy5amineAGF1332A[img]/KindEditor_4.0.1/attached/image/20130704/20130704110715_7750.jpg[/img]相似系列产品：
抗体、核酸、蛋白质等生物分子标记染料
羰基活性荧光染料
巯基反应性染料
羧酸类染料
产品主要应用：点击化学(Clickchemistry)、蛋白质组学研究中的双向荧光差异凝胶电泳(2DDIGE)和实时荧光定量PCR(RealtimePCR)。
菁染料是性能优良的荧光标记染料，摩尔吸光系数在荧光染料中是最高的。N-羟基琥珀酰亚胺酯是最常用的脂肪氨基标记试剂，广泛用于蛋白质、氨基酸多肽、抗体、核酸及其他生物分子的标记和检测。通过改变次甲基链的长度,可改变其荧光发射波长，每增加一个双键，按照Huoffman规则正好红移约100nm。
菁染料Cy3和Cy5已成为基因芯片的首选荧光标记物；另外,Cy5,Cy5.5和Cy7，Cy7.5的吸收在近红外区背景非常低，是荧光强度最高、最稳定的长波长染料，特别适合于活体小动物体内成像。但由于菁染料,尤其是不对称菁染料的合成副反应多,副产物极性相近,产物的分离提纯相当困难。菁染料特别是水溶性菁染料分子极性大,分离提纯越加困难。Lumiprobe供应脂溶性和水溶性菁染料。
相关产品：
产品分子结构可替代染料编号：AGF1371A
6-ROX-N-羟基琥珀酰亚胺酯
ROXNHSester,pure6-isomer[img]/KindEditor_4.0.1/attached/image/20130704/20130704102819_7906.jpg[/img]AlexaFluor568编号：AGF1326A
Cy3-N-羟基琥珀酰亚胺酯
Cy3NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704103024_8531.jpg[/img]AlexaFluor546NHSester
DyLight549NHSester
编号：AGF1330A
Cy3.5-N-羟基琥珀酰亚胺酯
Cy3.5NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704103242_2437.jpg[/img]AlexaFluor594NHSester
DyLight594NHSester
编号：AGF1338A
Cy5-N-羟基琥珀酰亚胺酯
Cy5NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704103422_4468.jpg[/img]AlexaFluor647NHSester
DyLight649NHSester
编号：AGF1345A
Cy5.5-N-羟基琥珀酰亚胺酯
Cy5.5NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704103557_5875.jpg[/img]AlexaFluor680NHSester
DyLight680NHSester
编号：AGF1349A
Cy7-N-羟基琥珀酰亚胺酯
Cy7NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704103713_8843.jpg[/img]编号：AGF1356A
Cy7.5-N-羟基琥珀酰亚胺酯
Cy7.5NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704103756_5406.jpg[/img]编号：AGF1374A
磺酸基-Cy3-N-羟基琥珀酰亚胺酯
Sulfo-Cy3NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704103830_1656.jpg[/img]AlexaFluor546
DyLight549
编号：AGF1377A
磺酸基-Cy5-N-羟基琥珀酰亚胺酯
Sulfo-Cy5NHSester[img]/KindEditor_4.0.1/attached/image/20130705/20130705095752_6656.jpg[/img]AlexaFluor647
DyLight649
编号：AGF1379A
磺酸基-Cy7-N-羟基琥珀酰亚胺酯
Sulfo-Cy7NHSester[img]/KindEditor_4.0.1/attached/image/20130704/20130704104047_6968.jpg[/img]相似系列产品：
羰基活性荧光染料
氨基类染料
巯基反应性染料
羧酸类染料
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——高中唯一一个活体染色剂。染线粒体的，染成蓝绿色
暂无品牌问答
