Fluo-3, AM, Cell Permeant 鈣離子熒光探針，超級純

n   最常用的Ca²⁺綠色熒光探針

搜索關鍵詞：Fluo-3 AM, Cell Permeant 鈣離子熒光探針，超級純；Fluo-3, Acetoxymethyl Ester; 可見光激發波長Ca²⁺熒光探針；Fluo-4；Fluo-8, AM；Rhod-2, AM；CAS NO：121714-22-5；

Fluo-3是一種常用的可見光激發波長Ca²⁺熒光探針，具有較高的Ca²⁺捕獲能力，通過熒光強度的變化直接反映Ca²⁺信號的動力學變化。當Fluo-3以游離配體形式存在時幾乎是非熒光性的，但一旦與Ca²⁺結合即可產生很強的熒光，熒光信號增強60~80倍，此時的最大激發波長為506nm，最大發射波長為526nm?？赏ㄟ^激光共聚焦顯微鏡或流式細胞儀來檢測胞內鈣離子水平的變化。Fluo-3, AM是Fluo-3的一種乙酰甲酯衍生物，具有細胞膜滲透性，只需簡單培養，即可輕易進入細胞。一旦進入細胞內，即被其內酯酶剪切生成不具膜滲透性的Fluo-3，從而滯留在胞內以發揮相應生理功能。

可見光激發Ca²⁺熒光探針的優勢：

與紫外光激發的熒光探針相比，如Fura-2和Indo-1，可見光激發Ca²⁺探針具有以下特點：

1）適用于大多數的激光共聚焦測鈣系統，包括共聚焦激發掃描顯微鏡以及流式細胞儀等。

2） 具有更強的染料吸收性能，使得更低濃度的探針即可成功檢測Ca²⁺變化，從而降低對活細胞的光毒性。

3）Ca²⁺依賴性熒光強度增強，對Ca²⁺變化水平檢測敏感度更高。

4）降低樣品自熒光以及光散射的干擾。

5）兼容光激活探針以及UV-激發試劑，因此更方便于多參數檢測。

6）無光譜偏移。

Fluo-3, AM的應用：

Fluo-3自1989年發現之后，其成像技術常用來揭示鈣離子信號傳遞內許多元素發展過程的空間動力學。還能被廣泛用在流式分析，比如籠鎖Ca²⁺螯合劑光活化、第二信使、神經傳導物質研究以及細胞水平的藥物篩選實驗。

Fluo-3, AM的應用實例：

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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