钙离子荧光探针Fluo2 AM

钙测量对于许多生物学研究至关重要。钙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钙浓度的变化。 Fluo-2 是 Fl​​uo-3 和 Fluo-4 的母体化合物。这些荧光钙指示剂具有钙依赖性荧光。

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-2 AM中储备溶液。

工作溶液配制

Fluo-2 AM工作溶液
1.在实验当天，将Fluo-2AM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-2 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-2 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-2 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-2 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。

试剂应用文献

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Discrimination of Dormant and Active Hematopoietic Stem Cells by G0 Marker Reveals Dormancy Regulation by Cytoplasmic Calcium
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Ketamine Increases Proliferation of Human iPSC-Derived Neuronal Progenitor Cells via Insulin-Like Growth Factor 2 and Independent of the NMDA Receptor
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P2Y6 signaling in alveolar macrophages prevents leukotriene-dependent type 2 allergic lung inflammation
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TRPA1-dependent reversible opening of tight junction by natural compounds with an $\alpha$, $\beta$-unsaturated moiety and capsaicin
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A new electro-optical approach for conductance measurement: an assay for the study of drugs acting on ligand-gated ion channels
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Altered spontaneous calcium signaling of in situ chondrocytes in human osteoarthritic cartilage
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Journal: Scientific reports (2017): 17093

Bystander effects elicited by single-cell photo-oxidative blue-light stimulation in retinal pigment epithelium cell networks
Authors: Ishii, Masaaki and Rohrer, Bärbel
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Bystander effects elicited by single-cell photo-oxidative blue-light stimulation in retinal pigment epithelium cell networks
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High-throughput screen detects calcium signaling dysfunction in typical sporadic autism spectrum disorder
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参考文献

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