钙离子荧光探针Fluo8L, AM

钙测量对于许多生物学研究至关重要。结合 Ca2+ 时显示光谱响应的荧光探针使研究人员能够通过使用荧光显微镜、流式细胞术、荧光光谱和荧光酶标仪来研究细胞内游离 Ca2+ 浓度的变化。 Fluo-3 AM 和 Fluo-4 AM 是活细胞钙成像中最常用的可见光激发钙指示剂。然而，Fluo-3 AM 和 Fluo-4 AM 在酯酶水解后仅在活细胞中发出适度的荧光，并且需要严酷的细胞负载条件才能最大化其细胞钙反应。 Fluo-8® 染料的开发旨在改善细胞负载和钙响应，同时保持方便的 Fluo-3 和 Fluo-4 光谱波长 Ex/Em = ∼490/∼520 nm。 Fluo-8® AM 可以在室温下加载到细胞中，而 Fluo-3 AM 和 Fluo-4 AM 需要 37°C 才能加载细胞。此外，Fluo-8® AM 的亮度是 Fluo-4 AM 的两倍，是 Fluo-3 AM 的四倍。 AAT Bioquest 提供一套出色的 Fluo-8® 试剂，具有不同的钙结合亲和力（Fluo-8® Kd = 389 nM；Fluo-8H™ Kd = 232 nM；Fluo-8L™ Kd = 1.86 µM；Fluo-8FF ™ Kd = 10 µM）。我们还提供多种包装尺寸以满足您的特殊需求（例如 1 mg、10x50 µg、20x50 µg 和 HTS 包装），无需额外包装费用。百萤生物是AAT Bioquest 的中国代理商，为您提供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

实验方案

储备溶液配制

在高质量无水DMSO制备2至5 mM 的Fluo-8L AM中储备溶液。

工作溶液配制

Fluo-8L AM工作溶液
1.在实验当天，将Fluo-8LAM溶解在DMSO中，或将指示剂储备溶液的等分试样解冻至室温。

2.在含有0.04%Pluronic®F-127的缓冲液（例如Hanks和Hepes缓冲液）中制备2至20µM Fluo-8L AM工作溶液。对于大多数细胞系，建议使用最终浓度为4-5μM的Fluo-8L AM。细胞负载所需指示剂的确切浓度请根据经验确定。

注：非离子洗涤剂Pluronic F-127用于提高Fluo-8L 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 Fluo-8L AM工作溶液添加到孔板中。
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.将加入染料的孔板在37°C的细胞培养箱中培养30至60分钟。
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.根据需要添加刺激物，并同时使用配备有FITC滤波片组的荧光显微镜或包含可编程液体处理系统（如FDSS、FLIPR或FlexStation）的荧光酶标仪在490/525nm，Cutoff=515nm处检测荧光强度。

图示

将U2OS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，将细胞分别与100μLFluo-3 AM，Fluo-4 AM和Fluo-8 AM在HHBS中以4μM浓度在37℃，5％CO 2中温育。 孵化器1小时。 用200μLHHBS洗涤细胞两次，然后使用FITC通道用荧光显微镜（Olympus IX71）成像。

试剂应用文献

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参考文献

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