| Overview |
 Printer Friendly Version
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| Ex/Em (nm) | 492/514 |
| MW | 1004.17 |
| CAS # | N/A |
| Solvent | Water |
| Storage | F/D/L |
| Category |
Cell Biology pH and Ion Indicators |
| Related |
| Spectrum | Advanced Spectrum Viewer |
Use of Cal-520® AM, Cal-590™ AM, or Cal-630™ AM Esters
1. Load Cells with Cal-520®, Cal-590™ or Cal-630™ AM Esters:
AM esters are non-polar esters that can 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 cells noninvasively. However, cautions must be exercised 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 Cal-520® AM,Cal-590™ AM or Cal-630™ 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 Cal-520® AM, Cal-590™ AM or Cal-630™ AM esters in high-quality, anhydrous DMSO.
b) On the day of the experiment, either dissolve Cal-520® AM, Cal-590™ AM or Cal-630™ AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a dye working solution of 10 to 20 µM in Hanks and Hepes buffer (HHBS) or the buffer of your choice with 0.04% Pluronic® F-127. The exact concentration of the indicator required for cell loading must be determined empirically.
Note: The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Cal-520® AM, Cal-590™ AM or Cal-630™ AM esters. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.
c) If your cells (such as CHO cells) contain organic anion-transports, then probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 mM) 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 in a cell incubator for 60 to 90 minutes, and then incubate the plate at room temperature for another 30 minutes.
Note: Incubating the dye longer than 2 hours gives better signal intensity for some cell lines.
f) Replace the dye working solution with HHBS or if applicable, a buffer of your choice that contains an anion transporter inhibitor, such as 1 mM probenecid, to remove excess probes.
g) Run the experiments at Ex/Em = 490/525 nm (for Cal-520® AM), 540/590 nm (for Cal-590™ AM) or 600/640 nm (for Cal-630™ AM).
2. Measure Intracellular Calcium Responses:
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Figure 1. Response of endogenous P2Y receptor to ATP in CHO-M1 cells without probenecid. CHO-M1 cells were seeded overnight at 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. 100 µl of 4 µM Fluo-3 AM, Fluo-4 AM or Cal 520® AM in HHBS were added into the wells, and the cells were incubated at 37 °C for 2 hour. The dye loading medium were replaced with 100 µl HHBS, 50 µl of 300 µM ATP were added, and then imaged with a fluorescence microscope (Olympus IX71) using FITC channel.
A B
Figure 2. ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells measured with Cal-520® or Fluo-4 AM. CHO-K1cells were seeded overnight in 50,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. 100 µL of 5 µM Fluo-4 AM or the Cal-520® AM with (A) or without (B) 2.5 mM probenecid was added into the cells, and the cells were incubated at 37oC for 2 hours. ATP (50µL/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations.
Cal 590™ AM Cal 630™ AM
Control ATP Control ATP
Figure 3. Response of endogenous P2Y receptor to ATP in CHO-K cells. CHO-K cells were seeded overnight at 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. 100 µl of 4 µM Cal 590™ AM or Cal 630™ AM in HHBS with 1 mM probenecid were added into the wells, and the cells were incubated at 37 °C for 2 hour. The dye loading mediums were replaced with 100 µl HHBS and 1 mM probenecid , then imaged with a fluorescence microscope (Olympus IX71) using TRITC channel before and after adding 50 µl of 300 µM ATP.
| References & Citations |
 Citation Explorer
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Below, you may find a small sampling of specific Cal-520® AM applications. To inquire about a potential application of Cal-520® AM, or to consult with our fluorescent dye specialists, please contact us at support@aatbio.com or 1-800-990-8053.
In Neurobiology, Cal-520® AM has been used to study:
» Neuron single action potentials in neocortical neurons both in vitro and in vivo, and associated voltage-dependent calcium channels[1]
» Superior colliculus in mice in conjunction with two-photon calcium imaging to visualize retinal ganglion cells in the superficial lamina[2]
» Aldolase C compartments in mice, looking at complex spike synchrony and its relation to sensory processing in awake animals[3]
» Neural circuits in brain slice and whole brain preparation, specifically looking at individual action potentials in vivo[4]
» Ca2+ dependent cell signaling pathways using cultured human neuroblastoma SH-SY5Y cells and high-speed video-microscopy[5]
In Cell Signaling, Cal-520® AM has been used to study:
» Intracellular calcium in sperm using the microplate reader platform as a means to quantitating parameters such as motility[6]
» Calcium signaling pathways in meniscus fibrochondrocytes by way of visualizing calcium localization and concentration[7]
» Ca2+ mediated cellular signaling in relation to inositol triphosphate and its flux through gap junctions[8]
» Retinal wave-mediated formation of calcium transients in Müller glial cells with focus on expression of GCaMP3[9]
» Ca2+ signaling pathways in zebrafish sperm, specifically looking at calcium flux resulting from cGMP-induced hyperpolarization[10]
In Cardiology, Cal-520® AM has been used to study:
» Sarcoplasmic reticulum insofar as its role in cardiac excitation-contraction coupling and calcium spark events[11]
» Optical mapping of calcium in cardiac tissue slices to develop a framework for future investigations into calcium transients[12]
» Sodium-calcium exchanger functionality and mechanism with regards to burst pacemaker activity in knockout mice[13]
» Pacemaker modulation in embryonic heart as a function of inositol-1,4,5-triphosphate receptors[14]
» Calcium current and the role of potassium channel-interacting protein 2 (KChIP2) in mice with regards to heart failure[15]
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？
