鈣黃綠素AM是最流行的熒光探針之一，用于標(biāo)記和檢測(cè)活細(xì)胞的細(xì)胞功能。但是，鈣黃綠素AM的單色使得無(wú)法在多色應(yīng)用中使用這種有價(jià)值的試劑。例如，由于與GFP相同的顏色，不可能在結(jié)合GFP的細(xì)胞中使用鈣黃綠素AM。為了解決鈣黃綠素AM的顏色限制，我們開(kāi)發(fā)了鈣黃綠橙 ，鈣黃綠 和鈣黃綠 。這些新的Calcein AM類(lèi)似物與Calcein AM結(jié)合使用，可以對(duì)活細(xì)胞進(jìn)行多色標(biāo)記和功能分析。非熒光鈣黃綠素 AM可以輕松進(jìn)入活細(xì)胞并水解產(chǎn)生強(qiáng)熒光鈣黃綠素 染料。可以使用通用的TRITC / Cy3濾波片組檢測(cè)Calcein Red 染料。

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操作步驟

      AM酯是非極性酯，很容易穿過(guò)活細(xì)胞膜，并被活細(xì)胞內(nèi)的細(xì)胞酯酶迅速水解。AM酯被廣泛用于無(wú)創(chuàng)地將各種極性熒光探針裝載到活細(xì)胞中。但是，使用AM酯時(shí)必須特別注意，因?yàn)樗鼈円子谒猓貏e是在溶液中。在使用高質(zhì)量無(wú)水二甲基亞砜（DMSO）之前，應(yīng)將其重新配制。DMSO儲(chǔ)備溶液可在-20°C的環(huán)境中干燥保存，并避光。在這些條件下，AM酯應(yīng)穩(wěn)定數(shù)月。

以下是我們推薦的將AM酯加載到活細(xì)胞中的方案。該協(xié)議僅提供指南，應(yīng)根據(jù)您的特定需求進(jìn)行修改。

1.準(zhǔn)備2至5 mM的AM酯高品質(zhì)無(wú)水DMSO儲(chǔ)備溶液。非離子型洗滌劑F-127有時(shí)用于增加AM酯的水溶性。

注：20％的Pluronic等體積® F-127溶液可以稀釋到裝載緩沖器之前加入到細(xì)胞活力指標(biāo)DMSO儲(chǔ)備溶液。終的Pluronic ® F-127的濃度為約0.02％。

2.在實(shí)驗(yàn)當(dāng)天，將細(xì)胞活力的固體指示劑溶解在DMSO中，或?qū)⒅甘緞﹥?chǔ)備溶液的等分試樣解凍至室溫。在您選擇的緩沖液（例如Hanks和Hepes緩沖液）中準(zhǔn)備1至10 μM的工作溶液。對(duì)于大多數(shù)細(xì)胞系，建議終濃度為4至5 μM的細(xì)胞活力指標(biāo)。必須根據(jù)經(jīng)驗(yàn)確定細(xì)胞加載所需指示劑的確切濃度。

3.如果您的細(xì)胞中含有有機(jī)陰離子轉(zhuǎn)運(yùn)蛋白，則可將丙磺舒（1-2.5 mM）或亞砜并吡嗪（0.1-0.25 mM）添加到細(xì)胞培養(yǎng)基中，以減少去酯化指示劑的泄漏。在室溫或37°C下將細(xì)胞與AM酯孵育20分鐘至一小時(shí)。

注意：降低裝載溫度可能會(huì)減少指示器的分隔。

4.在無(wú)細(xì)胞活力指示劑的緩沖液（如果適用，包含陰離子轉(zhuǎn)運(yùn)蛋白抑制劑）中洗滌細(xì)胞，以去除多余的探針。

注意：立即通過(guò)流式細(xì)胞儀分析細(xì)胞樣品，以測(cè)定零時(shí)每個(gè)細(xì)胞的平均熒光強(qiáng)度，無(wú)需清洗。

5.在所需的Ex / Em波長(zhǎng)下運(yùn)行實(shí)驗(yàn)。

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