Overview |
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Ex/Em (nm) | 494/517 |
MW | 1046.93 |
CAS # | 1345980-40-6 |
Solvent | DMSO |
Storage | F/D/L |
Category |
GPCR Calcium GPCR Assays |
Related |
Calcium Channels pH and Ion Indicators |
- Enhanced Intensity → 2x brighter than Fluo-4 AM
- Better & Faster Loading → Room temperature instead of 37°C
- More Robust Results → Probe effective in a wide range of temperatures
- Variety of Kd → Many options for calcium binding dissociation constant
The History
With hundreds of thousands of peer-reviewed papers published annually, it is clear that there is both intense and widespread interest in calcium and its function in biological systems. This is because calcium ions (Ca2+) play a crucial role in a number of biological processes, ranging from signal transduction to muscle contraction to neurotransmitter regulation. This ubiquity of calcium, and the subsequent need to study it, has prompted extensive effort into the development of stable, sensitive and convenient calcium indicators, as researchers looked for tools to help quantitate and visualize intracellular calcium.
The first big breakthrough came in the 1980s, when a group of researchers at UC Berkeley developed a calcium indicator based on fluorescein. Their research laid the groundwork for now well-established calcium probes such as Fluo-3 AM and Fluo-4 AM. These fluorescein-based AM esters readily pass through the membranes of living cells, where once inside, they are rapidly hydrolyzed by esterases and set to detect calcium. Upon successful binding with calcium, Fluo-3 or Fluo-4 will become fluorescent, emitting a signal at ~520 nm when excited by a ~490 nm light source.
While a significant achievement, the first generation of fluorescein-based dyes suffered from several major problems, specifically with loading conditions and brightness. Cells had to be treated with the fluorescein-based AM ester under harsh conditions that could damage the integrity of the sample. And if loading was not conducted under just perfect conditions, signal quality and intensity would be dramatically diminished. Such prominent deficiencies should not exist in a fluorescent probe, particularly one investigating a substance as vital to biology as calcium. It is with this thought in mind that AAT Bioquest set out to develop a new fluorescein-based calcium indicator. And after much research, development and optimization, we are pleased to announce the next generation of fluorescein-based dyes, headed by our flagship indicator, Fluo-8® AM.
Fluo-8® AM Features
Fluo-8® AM is a novel fluorescent green calcium indicator that builds upon the fluorescein core while rectifying the issues that plagued earlier indicators like Fluo-3 AM and Fluo-4 AM. In particular, it has several key advantages:
- Enhanced Intensity - Tests have shown Fluo-8® AM is twice as bright as Fluo-4 AM and up to four times brighter than Fluo-3 AM
- Better Loading - Fluo-8® AM can be loaded at room temperature, unlike the 37°C incubation required by Fluo-4 AM
- Faster Loading - Room temperature loading means no need to warm up and cool down cells; finish loading in as little as 20 minutes
- Robust - Fluo-8® is less temperature dependent than other probes, leading to more consistent results
- Versatile Kd - Fluo-8® AM is available with a variety of Ca2+ binding dissociation constants, see Table 1 for details
As with its predecessors, Fluo-8® AM is completely compatible with use in flow cytometry, fluorescence microscopy, fluorescence spectroscopy and the fluorescence microplate platform. To read more about how Fluo-8® AM has revolutionized calcium imaging, or to see additional Fluo-8® product applications, please click here.
Table 1. Ca2+ Binding Dissociation Constants
Product | Excitation | Emission | Ca2+ Kd | Cat No. | Pricing |
Fluo-8® AM | 490 nm | 514 nm | 389 nM | 21080 | $245 for 1 mg |
Fluo-8H™ AM | 490 nm | 514 nm | 232 nM | 21090 | $295 for 1 mg |
Fluo-8L™ AM | 490 nm | 514 nm | 1.86 µM | 21096 | $295 for 1 mg |
Fluo-FF™ AM | 494 nm | 514 nm | 10 µM | 21105 | $295 for 1 mg |
Spectrum | Advanced Spectrum Viewer |
Use of Fluo-8® AM Esters
1. Load Cells with Fluo-8® AM Esters:
AM esters are the non-polar esters that readily cross live cell membranes, and rapidly hydrolyzed by cellular esterases inside live cells. AM esters are widely used for loading a variety of polar fluorescent probes into live cell non-invasively. However, cautions must be excised when AM esters are used since they are susceptible to hydrolysis, particularly in solution. They should be reconstituted just before use in high-quality, anhydrous dimethylsulfoxide (DMSO). DMSO stock solutions may be stored desiccated at –20 °C and protected from light. Under these conditions, AM esters should be stable for several months.
Following is our recommended protocol for loading Fluo-8® AM esters into live cells. This protocol only provides a guideline, and should be modified according to your specific needs.
a) Prepare a 2 to 5 mM stock solution of Fluo-8® AM esters in high-quality, anhydrous DMSO.
b) On the day of the experiment, either dissolve Fluo-8® in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a working solution of 1 to 10 µM in Hanks and Hepes buffer (HHBS) or the buffer of your choice with 0.02% Pluronic® F-127. For most of cell lines, Fluo-8® reagents with a concentration ranging from 4-5 uM are recommended. The exact concentration of the indicator required for cell loading must be determined empirically. To avoid any artifacts caused by overloading and potential dye toxicity, it is recommended to use the minimal dye concentration that can generate sufficient signal strength.
Note: The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Fluo-8® AM esters. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.
c) If your cells containing the organic anion-transports, probenecid (1–2.5 mM) or sulfinpyrazone (0.1–0.25 mM) may be added to the cell medium to reduce leakage of the de-esterified indicators.
Note: A variety of ReadiUse™ probenecid including water soluble sodium salt and stabilized solution can be purchased from AAT Bioquest.
d) Add equal volume of the dye working solution (from Step b or c) into your cell plate.
e) Incubate the dye-loading plate at a cell incubator or room temperature for 20 minutes to one hour.
Note: Decreasing the loading temperature might reduce the compartmentalization of the indictor.
f) Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 2.5 mM probenecid, if applicable) to remove excess probes.
g) Run the experiments at Ex/Em = 490/525 nm
Use of Screen Quest™ Fluo-8 NW Calcium Assay Kits for HTS Applications
GPCR activation can be detected by direct measurement of the receptor mediated cAMP accumulation, or changes in intracellular Ca2+ concentration. GPCR targets that couple via Gq produce an increase in intracellular Ca2+ that can be measured using a combination of Fluo-8® reagents and a fluorescence microplate reader. The fluorescence imaging plate readers (such as, FLIPR™, FDSS or BMG NovoStar™) have a cooled CCD camera imaging system which collects the signal from each well of a microplate (both 96 and 384-well) simultaneously. These plate readers can read at sub-second intervals, which enables the kinetics of the response to be captured, and has an integrated pipettor that may be programmed for successive liquid additions. Besides their robust applications for GPCR targets, our Screen Quest™ Fluo-8 Calcium Assay Kits can be also used for characterizing calcium ion channels and screening calcium ion channel-targeted compounds.
Figure 2. Carbachol Dose Response was measured in HEK-293 cells with Screen Quest™ Fluo-8 NW Assay kit and Fluo-4 NW Assay Kit. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with, respectively, 100 µL of the Screen Quest™ Fluo 8-NW calcium assay kit and Fluo-4 NW kit (according to the manufacturer’s instructions) for 1 hour at room temperature. Carbachol (25µL/well) was added by NOVOstar (BMG LabTech) to achieve the final indicated concentrations. The EC50 of Fluo-8 NW is about 1.2 uM.
Compared to other commercial calcium assay kits that either based on Fluo-3 or Fluo-4, our Screen Quest™ Calcium Assay Kits have the following advantages for HTS applications:
•Broad Applications: work with both GPCR and calcium channel targets.
•Convenient Spectral Wavelengths: maximum excitation @ ~490 nm; maximum emission @ ~514 nm.
•Flexible Dye Loading: dye loading at room temperature (rather than 37 ºC required for Fluo-4 AM).
•No Wash Required and No Quencher Interference with Your Targets.
•Robust Performance: enable calcium assays that are impossible with Fluo-4 AM or Fluo-3 AM.
•Strongest Signal Intensity: 2 times brighter than that of Fluo-4 AM; 4 times brighter than that of Fluo-3 AM.
Use of Fluo-8® Salts
Calcium calibration can be carried out by measuring the fluorescence intensity of the salt form (25 to 50 µM in fluorescence microplate readers) of the indicators in solutions with precisely known free Ca2+ concentrations. Calibration solutions can be used based on 30 mM MOPS EGTA Ca2+ buffer. In general, water contains trace amount of calcium ion. It is highly recommended to use 30 mM MOPS + 100 mM KCl, pH 7.2 as buffer system. One can simply make a 0 and 39 µM calcium stock solutions as listed below, and these 2 solutions are used to make a serial solution of different Ca2+ concentrations
A. 0 µM calcium: 30 mM MOPS + 100 mM KCl, pH 7.2 buffer + 10 mM EGTA
B. 39 µM calcium: 30 mM MOPS + 100 mM KCl, pH 7.2 buffer + 10 mM EGTA + 10 mM CaCl2
To determine either the free calcium concentration of a solution or the Kd of a single-wavelength calcium indicator, the following equation is used:
[Ca]free = Kd[F ─ Fmin]/Fmax ─ F]
Where F is the fluorescence intensity of the indicator at a specific experimental calcium level, Fmin is the fluorescence intensity in the absence of calcium and Fmax is the fluorescence intensity of the calcium-saturated probe.
The dissociation constant (Kd) is a measure of the affinity of the probe for calcium. The calcium-binding and spectroscopic properties of fluorescent indicators vary quite significantly in cellular environments compared to calibration solutions. In situ response calibrations of intracellular indicators typically yield Kd values significantly higher than in vitro determinations. In situ calibrations are performed by exposing loaded cells to controlled Ca2+ buffers in the presence of ionophores such as A-23187, 4-bromo A-23187 and ionomycin. Alternatively, cell permeabilization agents such as digitonin or Triton® X-100 can be used to expose the indicator to the controlled Ca2+ levels of the extracellular medium. The Kd values of Fluo-8® reagents are listed in Table 1 for your reference.
References & Citations |
Citation Explorer
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Below, you may find a small sampling of specific Fluo-8® AM applications sorted by field of study. To inquire about a potential application of Fluo-8® AM, or to consult with our fluorescent dye specialists, please contact us at support@aatbio.com or 1-800-990-8053.
In Oncology, Fluo-8® AM has been used to study:
» Breast cancer cells by monitoring intracellular Ca2+ flux associated with apoptosis and inhibition by 2-aminoethoxydiphenyl borate[1]
» Antitumor activity by way of thioredoxin-binding protein 2 and its dependence on intracellular calcium concentration[2]
» Bcl-1 and Bcl-2 regulation through characterization of cytosolic transport as quantified by calcium flux[3]
» Ca2+ influx and Ca2+ channel activity in NCI-H460 cells as a parameter for monitoring progression of non-small cell lung cancer[4]
» Ca2+ release by HN4 cells and CLIC4 upregulation of apoptosis through mitochondrial and endoplasmic reticulum pathways[5]
In Cardiology, Fluo-8® AM has been used to study:
» Low-energy far-field stimulation as a therapy for tachycardia and fibrillation[6]
» Calcium flux during calcium sparks in ventricular myocytes[7]
» Cardiac conduction as a function of cell rigidity in the context of cardiovascular disease[8]
» Diastolic Ca2+ transients in cardiac myocytes and SR-luminal and free cytoplasmic Ca2+ concentrations[9]
» Sphingosine-1-phosphate (S1P) receptor activation in valvular interstitial cells as detected by cytosolic Ca2+ flux[10]
In Neurobiology, Fluo-8® AM has been used to study:
» Hippocampal CA1 neurons, visualizating neurons to investigate the role of amyloid-β in the progression of Alzheimer's disease[11]
» Cytosolic Ca2+ concentrations in HEK293 cells and its regulatory effect on Aβ1-42 and hAmylin and associated signaling pathways [12]
» G protein-coupled receptors (GPRs) in response to cannabinoids in presynaptic CA3 or postsynaptic CA1 pyramidal cells [13]
» Medullary interneurons and dendritic calcium activity in regards to inspiratory bursts[14]
» N2a cell activation by histamine, as monitored by increases in intracellular Ca2+ concentrations[15]
In Stem Cells, Development & Differentiation, Fluo-8® AM has been used to study:
» Induction of pluripotent stem cells (iPSCs) into functioning cardiac cells, as validated by Ca2+ flux and membrane potential[16]
» CXCR4 and CXCR7 receptors in T cells and their role in cell survival and chemotaxis [17]
» Ca2+ uptake by myocytes derived from human induced pluripotent stem cells during pathogenesis of Duchenne muscular dystrophy[18]
» Agonist-induced calcium transients in differentiation of rat bone marrow mesenchymal stem cells into smooth muscle cells[19]
» Calcium channel blockades and their effect on cardiac progenitor cell proliferation and differentiation[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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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。
分子生物学实验的染料主要涉及到核酸染料和蛋白质染料.核酸染料主要有EB(溴化乙锭,高致癌性),goldview,sybr green(实时定量PCR时常用染料).这些染料可以和核酸双链分子特异性结合发出强荧光而被检测到.蛋白质染料最常用的是考马斯亮蓝 R-250,硝酸银.其中硝酸银有时也用于核酸染色.
染料分为天然染料和人工染料两种。天然染料有胭脂虫红、地衣素、石蕊和苏木素等,它们多从植物体中提取得到,其成分复杂,有些至今还未搞清楚。目前主要采用人工染料,也称煤焦油染料,多从煤焦油中提取获得,是苯的衍生物。多数染料为带色的有机酸或碱类,难溶于水,而易溶于有机溶剂中。为使它们易溶于水,通常制成盐类。
染料可按其电离后染料离子所带电荷的性质,分为酸性染料、碱性染料、中性(复合)染料和单纯染料四大类。 标本干燥后即进行固定,固定的目的有三个:
1)杀死微生物,固定细胞结构。
2)保证菌体能更牢的粘附在载玻片上,防止标本被水冲洗掉。
3)改变染料对细胞的通透性,因为死的原生质比活的原生质易于染色。
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。
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)
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?
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:)
请各位大侠给予帮助!!
谢谢!!
健那绿——高中唯一一个活体染色剂。染线粒体的,染成蓝绿色
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?
暂无品牌问答