Fluo-3, AM, Cell Permeant 鈣離子熒光(guāng)探針，超級純

n   最常用的Ca²⁺綠色熒光(guāng)探針

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

Fluo-3是一(yī)種常用的可見(jiàn)光(guāng)激發波長(cháng)Ca²⁺熒光(guāng)探針，具有較高(gāo)的Ca²⁺捕獲能(néng)力，通(tōng)過熒光(guāng)強度的變化直接反映Ca²⁺信号的動力學變化。當Fluo-3以遊離配體形式存在時幾乎是非熒光(guāng)性的，但一(yī)旦與Ca²⁺結合即可産生(shēng)很強的熒光(guāng)，熒光(guāng)信号增強60~80倍，此時的最大激發波長(cháng)為(wèi)506nm，最大發射波長(cháng)為(wèi)526nm。可通(tōng)過激光(guāng)共聚焦顯微鏡或流式細胞儀來檢測胞内鈣離子水(shuǐ)平的變化。Fluo-3, AM是Fluo-3的一(yī)種乙酰甲酯衍生(shēng)物(wù)，具有細胞膜滲透性，隻需簡單培養，即可輕易進入細胞。一(yī)旦進入細胞内，即被其内酯酶剪切生(shēng)成不具膜滲透性的Fluo-3，從(cóng)而滞留在胞内以發揮相(xiàng)應生(shēng)理功能(néng)。

可見(jiàn)光(guāng)激發Ca²⁺熒光(guāng)探針的優勢：

與紫外光(guāng)激發的熒光(guāng)探針相(xiàng)比，如Fura-2和Indo-1，可見(jiàn)光(guāng)激發Ca²⁺探針具有以下(xià)特點：

1）适用于大多(duō)數的激光(guāng)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(néng)，使得更低(dī)濃度的探針即可成功檢測Ca²⁺變化，從(cóng)而降低(dī)對活細胞的光(guāng)毒性。

3）Ca²⁺依賴性熒光(guāng)強度增強，對Ca²⁺變化水(shuǐ)平檢測敏感度更高(gāo)。

4）降低(dī)樣品自(zì)熒光(guāng)以及光(guāng)散射的幹擾。

5）兼容光(guāng)激活探針以及UV-激發試劑，因此更方便于多(duō)參數檢測。

6）無光(guāng)譜偏移。

Fluo-3, AM的應用：

Fluo-3自(zì)1989年(nián)發現之後，其成像技(jì)術(shù)常用來揭示鈣離子信号傳遞内許多(duō)元素發展過程的空間動力學。還(hái)能(néng)被廣泛用在流式分析，比如籠鎖Ca²⁺螯合劑光(guāng)活化、第二信使、神經傳導物(wù)質研究以及細胞水(shuǐ)平的藥物(wù)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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