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(guān)鍵詞：Fluo-3 AM, Cell Permeant 鈣離子熒光探針，超級純；Fluo-3, Acetoxymethyl Ester; 可見(jiàn)光激發(fā)波長(cháng)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(jiàn)光激發(fā)波長(cháng)Ca²⁺熒光探針，具有較高的Ca²⁺捕獲能力，通過(guò)熒光強度的變化直接反映Ca²⁺信號的動(dòng)力學(xué)變化。當Fluo-3以游離配體形式存在時(shí)幾乎是非熒光性的，但一旦與Ca²⁺結合即可產(chǎn)生很強的熒光，熒光信號增強60~80倍，此時(shí)的最大激發(fā)波長(cháng)為506nm，最大發(fā)射波長(cháng)為526nm?？赏ㄟ^(guò)激光共聚焦顯微鏡或流式細胞儀來(lái)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(jiǎn)單培養，即可輕易進(jìn)入細胞。一旦進(jìn)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(fā)揮相應生理功能。

可見(jiàn)光激發(fā)Ca²⁺熒光探針的優(yōu)勢：

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

1）適用于大多數的激光共聚焦測鈣系統，包括共聚焦激發(fā)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(lài)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(fā)試劑，因此更方便于多參數檢測。

6）無(wú)光譜偏移。

Fluo-3, AM的應用：

Fluo-3自1989年發(fā)現之后，其成像技術(shù)常用來(lái)揭示鈣離子信號傳遞內許多元素發(fā)展過(guò)程的空間動(dòng)力學(xué)。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(jīng)傳導物質(zhì)研究以及細胞水平的藥物篩選實(shí)驗。

Fluo-3, AM的應用實(shí)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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