实验方案
溶液配制
1.储备溶液配制
Calbryte 520 AM 储备溶液
在无水 DMSO 中制备 2 至 5 mM Calbryte 520 AM 储备溶液。
注意：当在 DMSO 中复溶时，Calbryte 520 AM 是透明、无色的溶液。
2.工作溶液配制
Calbryte 520 AM 工作溶液
2.1实验当天，将 Calbryte 520 AM 溶解在 DMSO 中，或将等份指示剂储备溶液解冻至室温。
2.2在您选择的缓冲液（例如 Hanks 和 Hepes 缓冲液）（货号20011）中用 0.04% Pluronic® F-127 制备 2 至 20 µM Calbryte 520 AM 工作溶液。对于大多数细胞系，建议终浓度为 4-5 μM 的 Calbryte 520 AM。细胞加载所需指示剂的准确浓度必须根据经验确定。
注意：非离子洗涤剂 Pluronic® F-127 有时用于增加 Calbryte 520 AM 的水溶性。可以从金畔购买购买。
注意：如果您的细胞含有有机阴离子转运蛋白，可将丙磺舒 (1-2 mM) 添加到染料工作溶液中（孔中的最终浓度为 0.5-1 mM），以减少脱酯后染料的外漏。各种 ReadiUse 丙磺舒产品，包括水溶性、钠盐和稳定溶液，均可从金畔购买。
操作步骤
以下是我们推荐的将Calbryte 520 AM加入活细胞的方案。 该方案仅提供指南，实际应根据您的特定需求进行修改。
a）将细胞在生长培养基中培养过夜。
b）第二天，将 1X Calbryte 520 AM 工作溶液添加到细胞孔板中。
注意：如果您的化合物干扰血清，请在上样前用新鲜的 HHBS 缓冲液替换生长培养基。
c）将载有染料的孔板在细胞培养箱中于 37°C 下孵育 30 至 60 分钟。
注意：孵育染料超过 1 小时可以提高某些细胞系的信号强度。
d）用 HHBS 或您选择的缓冲液（包含阴离子转运蛋白抑制剂，例如 1 mM 丙磺舒，如果适用）替换染料工作溶液，以去除任何多余的探针。
e）将染料加载板在细胞培养箱中孵育约60分钟，然后将板在室温下再孵育15分钟。
f）用HHBS或您选择的含有阴离子转运蛋白抑制剂（如1 mM丙磺舒）的缓冲液替换染料工作溶液，以去除多余的探针。
g）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）的荧光酶标仪在 Ex/Em = 490/525 nm 处测量荧光。
产品应用文献
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相关产品
| 产品名称 |
货号 |
| 钙离子荧光探针Cal-520 , AM |
Cat#21130 |
| 钙离子荧光探针Fluo-8, AM |
Cat#21080 |
| 新型钙离子荧光探针Calbryte 590, AM *细胞渗透性* |
Cat#20700 |
