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钙离子荧光探针Cal520FF, AM

英文名称:Cal-520FF™, AM
产品参数
Ex (nm)492Em (nm)515
分子量1138.92溶剂DMSO
存储条件在零下15度以下保存, 避免光照
产品概述

Cal-520 提供了一种强大的均质荧光检测工具,用于检测细胞内钙动员。 Cal-520 AM 是一种新型荧光钙敏感染料,与现有的绿色钙指示剂(如 Fluo-3 AM 和 Fluo-4 AM)相比,信噪比和细胞内保留能力显着提高。表达通过钙发出信号的 GPCR 或钙通道的细胞可以预加载可以穿过细胞膜的 Cal-520 AM。一旦进入细胞,Cal-520™AM 的亲脂性封闭基团就会被酯酶裂解,从而产生带负电荷的荧光染料,保留在细胞内。与钙结合后其荧光大大增强。当细胞受到激动剂刺激时,受体会发出细胞内钙释放的信号,从而显着增加 Cal-520® 的荧光。 Cal-520® AM 波长长、灵敏度高、荧光增强>100 倍等特点,使其成为细胞钙测量的理想指示剂。高信噪比和更好的细胞内保留使 Cal-520® 钙测定成为评估 GPCR 和钙通道靶标以及筛选其激动剂和拮抗剂的强大工具。与其他 Cal-520® 指示剂相比,Cal-520FF™ 对钙的亲和力最低,Kd ~ 10 uM。

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适用仪器


荧光显微镜  
Ex: FITC 滤波片组
Em: FITC 滤波片组
推荐孔板: 黑色透明底板

 


荧光酶标仪  
Ex: 490 nm
Em: 525 nm
Cutoff: 515 nm
推荐孔板: 黑色透明底板
读取模式: 底读模式/可分液处理
实验方案

实验方案

储备溶液配制

Cal-520FF AM 储备溶液

在高质量无水 DMSO 中制备 2 至 5 mM Cal-520FF AM 储备溶液。

注意:当在 DMSO 中复溶时,Cal-520FF™ AM 是透明、无色的溶液。

 

工作溶液配制

Cal-520FF AM 工作溶液
1.实验当天,将 Cal-520FF AM 溶解在 DMSO 中,或将等份指示剂储备溶液解冻至室温。

2.在您选择的缓冲液(例如 Hanks 和 Hepes 缓冲液)中用 0.04% Pluronic® F-127 制备 2 至 20 µM Cal-520FF™ AM 工作溶液。对于大多数细胞系,建议终浓度为 4-5 μM 的 Cal-520FF™ AM。细胞加载所需指示剂的准确浓度必须根据经验确定。

注:非离子洗涤剂Pluronic F-127用于提高Fluo-4 AM的水溶性。可从百萤购买各种Pluronic F-127溶液。
注:如果你的细胞含有有机阴离子转运蛋白,可以将丙磺舒(1-2mM)添加到染料工作溶液中(最终井内浓度为0.5-1mM),以减少酯化指示剂的泄漏。多种形式的丙磺舒产品,包括水溶性、钠盐和稳定溶液,可从百萤购买。

 

操作步骤

以下是我们推荐的将AM酯加载到活细胞中的方案。本方案仅提供指南,实际应根据您的具体需求进行修改。

1.在生长培养基中培养细胞过夜。
2.第二天,将 1X Cal-520FF™ AM 工作溶液添加到细胞板中。
注意:如果你的化合物干扰血清,在染色前用新鲜的HHBS缓冲液代替生长培养基。
3.将载有染料的板在细胞培养箱中于 37°C 下孵育 1 至 2 小时。
注:将染料培养2小时以上可以提高某些细胞系的信号强度。
4.用HHBS或您选择的缓冲液(含有阴离子转运蛋白抑制剂,如1mM丙磺舒)代替染料工作溶液,以去除任何多余的探针。
5.根据需要添加刺激剂,同时使用配备 FITC 滤光片组的荧光显微镜或包含可编程液体处理系统(例如 FDSS、FLIPR 或 FlexStation)的荧光板读数器在 Ex/Em = 490/525 nm 处测量荧光。

 

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