神經(jīng)末梢熒光探針

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SynaptoGreen C4 神經(jīng)末梢熒光探針 應(yīng)用：
※ SynaptoGreen C4已用于標(biāo)記EM網(wǎng)格上生長的培養(yǎng)神經(jīng)元，適用于熒光標(biāo)記、活體光學(xué)顯微鏡和成像研究等。
※ 一種適合于在突觸和神經(jīng)肌肉接頭處監(jiān)測突觸活動的熒光神經(jīng)末梢染料

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SynaptoGreen C4注意事項(xiàng)：
※ 保存和操作過程中注意避光
※ 在水溶液中基本無熒光，最大發(fā)射波長具pH依賴性。苯乙烯染料的光譜特征在甲醇或氯仿中測定。其在膜環(huán)境中的最大激發(fā)和最大發(fā)射波長都會變短。最大激發(fā)波長的差異通常在20nm，發(fā)射波長的差異通常是80nm，但依具體的探針有所差異。

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熒光光譜

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實(shí)驗(yàn)方法：
1. FM 1–43儲存液(1000X):用水溶解FM1-43配制4mM儲存液。按20μL每管分裝保存在-20℃避光。如果希望標(biāo)記后固定樣本，選擇FM 1–43。
2. HL-3 + 90 mM KCl溶液: 25 mM NaCl, 90 mM KCl, 10 mM NaHCO3, 5 mM HEPES,30 mM sucrose, 5 mM threalose, 10 mM MgCl2, pH to 7.2.新鮮制備，超過2d后不可使用，4℃保存。加入CaCl2（1M標(biāo)準(zhǔn)液）以得到終濃度為1.5mM的CaCl2（或希望使用的其他濃度）。

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工作液濃度：
選用預(yù)熱好的HBSS溶液，配制成 5-20 μM區(qū)間的FM 1-43 工作液。
1. 細(xì)胞染色(懸浮細(xì)胞染色)
1.1 通過離心收集細(xì)胞，然后加入 PBS 洗滌兩次，每次5分鐘。
細(xì)胞密度約在1×106/mL區(qū)間即可。
1.2 加入 1 mL FM 工作液，室溫孵育約5-30 分鐘。
1.3 400 g，離心 3-4 分鐘，棄去上清。
1.4 加入 PBS 洗滌細(xì)胞兩次，每次 5 分鐘。
1.5 用 1 mL 無血清培養(yǎng)基或 PBS 重懸細(xì)胞后，使用熒光顯微鏡或流式細(xì)胞儀進(jìn)行觀察。
2. 細(xì)胞染色(貼壁細(xì)胞染色)
2.1 將貼壁細(xì)胞培養(yǎng)于無菌蓋玻片上。
2.2 從培養(yǎng)基中移出蓋玻片，吸除多余培養(yǎng)基。
2.3 加入 100 μL 染料工作液，輕輕晃動使其完全覆蓋細(xì)胞，室溫孵育約5-30 分鐘。
2.4 吸去染料工作液，用培養(yǎng)基洗 2-3次，每次 5 分鐘 ，使用熒光顯微鏡或流式細(xì)胞儀進(jìn)行觀察。

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案例：

 

Live imaging shows that Piezo1 channels are functionally expressed on SGCs and neurons. (A). Fluorescent snapshots of 2DIV mouse trigeminal culture showing SGCs (blue arrows) and neurons (yellow arrows). The snapshots have been taken after 20 min of incubation with Fluo-4 AM. The left frame represents the trigeminal culture during baseline. The right frame shows cell activation after the application of 5 μM of Yoda1. Scale bar-50 μm. (B). Sample traces of calcium transients in trigeminal neurons induced by 5 μM of Yoda1 (n = 9). (C). Sample traces of calcium transients in trigeminal SGCs induced by 5 μM of Yoda1 (n = 12). (D). Statistics of the percentage of increased fluorescence intensity normalized to baseline in neurons (n = 9) vs. SGCs (n = 12) treated with 5 μM of Yoda1. There is no difference in Yoda1 responses among the different cell types (unpaired t-test, p = 0.120).

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FM1-43 remains trapped after application of the Piezo1 agonist Yoda1 in Piezo1-transfected HEK cells. (A). Fluorescence snapshots of Piezo1-transfected HEK cells after 1 min application of 2 μM of FM1-43 (1st frame), during washout following only FM1-43 application (second frame). The third frame is captured at the top of 2 μM of FM1-43 + 25 μM of Yoda1 treatment (transient peak), followed by washout (fourth frame), where the dye remained trapped (tail response). Calibration bar-10 μm. (B). Sample trace of fluorescence intensity variations induced by double application of 2 μM of FM1-43 and following 2 μM of FM1-43 + 25 μM of Yoda1 for Piezo1-transfected HEK cells. Important elements in order to understand the outcome of the experiment are the transient peak, showing a treatment-induced acute response, and the tail response, addressing the long-lasting dye internalization. (C). Statistics of the percentage of increased fluorescence intensity of the transient peak increase in different cells when 2 μM of FM1-43 + 25 μM of Yoda1 are applied, compared to only 2 μM of FM1-43 (paired t-test, *** p < 0.001). (D). Statistics of the percentage of increased fluorescence intensity of the tail response (baseline) increase in different cells when 2 μM of FM1-43 + 25 μM of Yoda1 are applied, compared to only 2 μM of FM1-43 (paired t-test, *** p < 0.001).