Fluo-3, AM, Cell Permeant 钙离子荧光探针，超级纯

n   最常用的Ca²⁺绿色荧光探针

搜索关键词：Fluo-3 AM, Cell Permeant 钙离子荧光探针，超级纯；Fluo-3, Acetoxymethyl Ester; 可见光激发波长Ca²⁺荧光探针；Fluo-4；Fluo-8, AM；Rhod-2, AM；CAS NO：121714-22-5；

Fluo-3是一种常用的可见光激发波长Ca²⁺荧光探针，具有较高的Ca²⁺捕获能力，通过荧光强度的变化直接反映Ca²⁺信号的动力学变化。当Fluo-3以游离配体形式存在时几乎是非荧光性的，但一旦与Ca²⁺结合即可产生很强的荧光，荧光信号增强60~80倍，此时的最大激发波长为506nm，最大发射波长为526nm。可通过激光共聚焦显微镜或流式细胞仪来检测胞内钙离子水平的变化。Fluo-3, AM是Fluo-3的一种乙酰甲酯衍生物，具有细胞膜渗透性，只需简单培养，即可轻易进入细胞。一旦进入细胞内，即被其内酯酶剪切生成不具膜渗透性的Fluo-3，从而滞留在胞内以发挥相应生理功能。

可见光激发Ca²⁺荧光探针的优势：

与紫外光激发的荧光探针相比，如Fura-2和Indo-1，可见光激发Ca²⁺探针具有以下特点：

1）适用于大多数的激光共聚焦测钙系统，包括共聚焦激发扫描显微镜以及流式细胞仪等。

2） 具有更强的染料吸收性能，使得更低浓度的探针即可成功检测Ca²⁺变化，从而降低对活细胞的光毒性。

3）Ca²⁺依赖性荧光强度增强，对Ca²⁺变化水平检测敏感度更高。

4）降低样品自荧光以及光散射的干扰。

5）兼容光激活探针以及UV-激发试剂，因此更方便于多参数检测。

6）无光谱偏移。

Fluo-3, AM的应用：

Fluo-3自1989年发现之后，其成像技术常用来揭示钙离子信号传递内许多元素发展过程的空间动力学。还能被广泛用在流式分析，比如笼锁Ca²⁺螯合剂光活化、第二信使、神经传导物质研究以及细胞水平的药物筛选实验。

Fluo-3, AM的应用实例：

Fig. 1, Ligand-induced changes in intracellular free [Ca²⁺]. Splenic B cells were incubated with 4μM Fluo-3,  AM at 5 x 10⁶ cells/ml and then adjusted to 7.5 x 10⁶ cells/ml in Hank's Balanced Salt Solution (HBSS) containing bovine serum albumin (BSA) for the assay. Anti-IgM (0.3 μM), SDF-1 (6 nM), LPA (1 μM), or vehicle was added at time 0. The assay was performed in a 96-well plate using a Fluoroskan Ascent Microplate Fluorometer.   Measurements were recorded for 10 min followed by sequential addition of NP-40 (0.5% final) and EGTA (45mM final) to obtain fluorescence maximum and minimum values, respectively. Intracellular free [Ca²⁺] was calculated using the equation: [free Ca²⁺] = Kd (F - Fmin)/(Fmax - F). The K_d for Fluo-3 was assumed to be 390 nM. Data was corrected for baseline drift caused by leakage of Fluo-3 AM from cells into Ca²⁺-containing medium.   Each graph shows tracings of three replicate samples from one experiment. Experiments were performed at least five times.

Fig. 2， Metal-ion response screening for fluo-3. The maximum relative fluorescence intensity was measured for identical indicator concentrations in solutions containing 10 mM EGTA + 10 µM TPEN, 1 µM ion (100 µM for Mg²⁺) and 100 µM ion (10 mM for Mg²⁺). Results are plotted as fluorescence changes relative to the ion-free (10 mM EGTA + 10 µM TPEN) reference solution expressed as (F-Fo)/Fo, where F is the fluorescence intensity of ion-containing solutions and Fo is the fluorescence intensity of the reference solution. Blue bars indicate the response to 1 µM ion (100 µM for Mg²⁺), and red bars indicate the response to 100 µM ion (10 mM for Mg²⁺).

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