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钙离子荧光探针Fluo-8，钠盐
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货号 | 21088 | 存储条件 | 在零下15度以下保存, 避免光照 |
| 规格 | 10×50 ug | 价格 | 2604 | |
| Ex (nm) | 495 | Em (nm) | 516 | |
| 分子量 | 796.53 | 溶剂 | Water | |
| 产品详细介绍 | ||||
简要概述
钙离子荧光探针Fluo-8，钠盐是美国AAT Bioquest生产的用于标记钙离子的荧光探针，在许多生物学研究中钙离子的测定非常重要。在与钙离子结合的条件下，荧光探针显现出光谱响应，这使研究者可以通过利用荧光显微镜、流式细胞仪、荧光分光镜和荧光分光仪等来研究细胞内部游离的Ca2+浓度的变化。在众多的被可见光激发的钙离子指示剂中，Fluo-3 和 Fluo-4是经常使用的。然而，Fluo-3 AM和Fluo-4 AM在酯酶水解作用下，在活细胞中只产生中度荧光；并且需要更苛刻的细胞载入条件使它们细胞的钙离子响应最大化。Quest Fluo-8 是在保持了Fluo-3和Fluo-4便捷地光谱波长，最大激发光在~490 nm处，最大发生光在~520 nm处的同时，提高了细胞载入和钙离子响应能力。Quest Fluo-8 AM只需要在室温下完成细胞载入，而Fluo-3 AM 和Fluo-4 AM需要在37℃的条件下。此外，Quest Fluo-8 的荧光亮度是Fluo-4 AM的两倍，是Fluo-3 AM的四倍。AAT Bioquest提供一组不同凡响的Quest Fluo-8 试剂，它们与钙离子的结合力都不相同(Quest Fluo-8: Kd = 389 nM; Quest Fluo-8H: Kd = 232 nM; Quest Fluo-8L: Kd = 1.86nM; Quest Fluo-8FF: Kd = 10 nM)。我们也提供多种不同大小的包装以满足你们的特殊要求，例如1 mg; 10×50 ug; 20×50 ug;高通量筛选大包装，不需要您做任何包装剂量上的改变，可直接用于HTS高通量筛选实验分析。金畔生物是AAT Bioquest 的中国代理商，为您提供最优质的钙离子荧光探针。
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钙离子篇：时间轴式讲解应用于钙离子检测的探针
产品说明书
操作步骤
使用Fluo-8®AM酯类
1.使用Fluo-8®AM酯：
AM酯是非极性酯，其易于穿过活细胞膜，并且通过活细胞内的细胞酯酶快速水解。AM酯广泛用于非侵入性地将各种极性荧光探针装载到活细胞中。但是，使用AM酯时必须小心，因为它们易于水解，特别是在溶液中。它们应在使用前重新配制成高质量的无水二甲基亚砜（DMSO）。DMSO储备溶液可以在-20℃下干燥储存并避光。在这些条件下，AM酯应稳定数月。
以下是我们推荐的将Fluo-8®AM酯加入活细胞的方案。该协议仅提供指南，应根据您的具体需求进行修改。
a）在高质量无水DMSO中制备2至5 mM Fluo-8®AM酯原液。
b）在实验当天，将Fluo-8® 溶解在DMSO中或将等份的指示剂储备溶液解冻至室温。在Hanks和Hepes缓冲液（HHBS）或0.02％Pluronic®F-127的缓冲液中制备1至10μM的工作溶液。对于大多数细胞系，建议使用浓度范围为4-5 uM的Fluo-8®试剂。细胞加载所需指示剂的确切浓度必须凭经验确定。为避免因过载和潜在染料毒性引起的任何伪影，建议使用可产生足够信号强度的最小染料浓度。
注意：非离子洗涤剂Pluronic®F-127有时用于增加Fluo-8®AM 酯的水溶性。
c）如果您的细胞含有有机阴离子转运蛋白，可以在细胞培养基中加入丙磺舒（1-2.5 mM）或磺吡酮（0.1-0.25 mM），以减少脱酯化指标的泄漏。
d）将等体积的染料工作溶液（来自步骤b或c）加入细胞板中。
e）在细胞培养箱中孵育或染料装载板室温下为20分钟至一小时。
f）用HHBS或您选择的缓冲液（含有阴离子转运蛋白抑制剂，如2.5 mM丙磺舒，如果适用）替换染料工作溶液，以去除多余的探针。
g）运行用Ex / Em比值=490/525纳米
使用Screen Quest Fluo-8 NW钙测定试剂盒进行HTS应用
可以通过直接测量受体介导的cAMP积累或细胞内Ca 2+浓度的变化来检测GPCR活化。通过Gq偶联的GPCR靶标产生细胞内Ca 2+的增加可以使用Fluo-8®试剂和荧光酶标仪的组合进行测量。荧光成像板读取器（例如，FLIPR TM，FDSS或BMG NovoStar ）具有冷却的CCD相机成像系统，其同时收集来自微孔板（96孔和384孔）的每个孔的信号。这些读板器可以以亚秒的间隔读取，这使得能够捕获响应的动力学，并且具有可以被编程用于连续液体添加的集成移液器。除了对GPCR靶标的强大应用外，我们的Screen Quest Fluo-8钙测定试剂盒还可用于表征钙离子通道和筛选钙离子通道靶向化合物。
图1.使用Screen Quest Fluo-8 NW测定试剂盒和Fluo-4 NW测定试剂盒在HEK-293细胞中测量卡巴胆碱剂量反应。 将HEK-293细胞以40,000个细胞/100μL/孔接种过夜，置于96孔黑色壁/透明底板中。除去生长培养基，并将细胞分别与100μL的Screen Quest Fluo 8-NW钙测定试剂盒和Fluo-4 NW试剂盒（根据制造商的说明书）在室温下温育1小时。通过NOVOstar（BMG LabTech）添加卡巴胆碱（25μL/孔）以达到最终指示的浓度。Fluo-8 NW 的EC 50约为1.2 uM。
与基于Fluo-3或Fluo-4的其他商业钙测定试剂盒相比，我们的Screen Quest 钙测定试剂盒具有以下HTS应用优势：
- 广泛的应用：与GPCR和钙通道目标一起使用。
- 方便的光谱波长：最大激发波长@ 490 nm; 最大发射@ ~514 nm。
- 灵活的染料加载：室温下的染料加载（而不是Fluo-4 AM所需的37ºC）。
- 无需清洗，无淬火干扰您的目标。
- 强大的性能：使用Fluo-4 AM或Fluo-3 AM无法进行钙分析。
- 最强信号强度：比Fluo-4 AM亮2倍;比Fluo-3 AM亮4倍。
使用Fluo-8® 盐
钙校准可以通过测量具有精确已知的游离Ca 2+浓度的溶液中的指示剂的盐形式（荧光酶标仪中25至50μM）的荧光强度来进行。可以基于30mM MOPS EGTA Ca 2+缓冲液使用校准溶液。通常，水含有微量的钙离子。强烈建议使用30 mM MOPS + 100 mM KCl，pH 7.2作为缓冲系统。可以简单地制备如下所列的0和39μM钙原液，这两种溶液用于制备不同Ca 2+浓度的连续溶液
A.0μM钙：30mM MOPS + 100mM KCl，pH 7.2缓冲液+ 10mM EGTA
B.39μM钙：30mM MOPS + 100mM KCl，pH 7.2缓冲液+ 10mM EGTA + 10mM CaCl 2
为了确定溶液的游离钙浓度或 单波长钙指示剂的K d，使用以下等式：
的[Ca] 游离 = K d [F─˚F 分钟 ] / F 最大 ─F]
其中F是特定实验钙水平下指示剂的荧光强度，F min是不存在钙时的荧光强度，F max是钙饱和探针的荧光强度。
解离常数（K d）是探针对钙的亲和力的量度。与校准溶液相比，荧光指示剂的钙结合和光谱性质在细胞环境中变化非常显着。细胞内指标的原位反应校准通常产生显着高于体外测定的K d值。通过在离子载体如A-23187,4-溴A-23187和离子霉素存在下将加载的细胞暴露于受控的Ca 2+缓冲液来进行原位校准。或者，细胞透化剂如洋地黄皂苷或Triton®X-100可用于将指示剂暴露于受控Ca.2+水平的细胞外培养基。
试剂应用文献
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Altered spontaneous calcium signaling of in situ chondrocytes in human osteoarthritic cartilage
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Analysis of Ca2+ response of osteocyte network by three-dimensional time-lapse imaging in living bone
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