鈣黃綠素AM是最流行的熒光探針之一，用于標記和檢測活細胞的細胞功能。但是，鈣黃綠素AM的單色使得無法在多色應用中使用這種有價值的試劑。例如，由于與GFP相同的顏色，不可能在結合GFP的細胞中使用鈣黃綠素AM。為了解決鈣黃綠素AM的顏色限制，我們開發了鈣黃綠橙 ，鈣黃綠 和鈣黃綠 。這些新的Calcein AM類似物與Calcein AM結合使用，可以對活細胞進行多色標記和功能分析。非熒光鈣黃綠素 AM可以輕松進入活細胞并水解產生強熒光鈣黃綠素 染料?？梢允褂猛ㄓ玫腡RITC / Cy3濾波片組檢測Calcein Red 染料。

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操作步驟

      AM酯是非極性酯，很容易穿過活細胞膜，并被活細胞內的細胞酯酶迅速水解。AM酯被廣泛用于無創地將各種極性熒光探針裝載到活細胞中。但是，使用AM酯時必須特別注意，因為它們易于水解，特別是在溶液中。在使用高質量無水二甲基亞砜（DMSO）之前，應將其重新配制。DMSO儲備溶液可在-20°C的環境中干燥保存，并避光。在這些條件下，AM酯應穩定數月。

以下是我們推薦的將AM酯加載到活細胞中的方案。該協議僅提供指南，應根據您的特定需求進行修改。

1.準備2至5 mM的AM酯高品質無水DMSO儲備溶液。非離子型洗滌劑F-127有時用于增加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胞活力指標DMSO儲備溶液。終的Pluronic ® F-127的濃度為約0.02％。

2.在實驗當天，將細胞活力的固體指示劑溶解在DMSO中，或將指示劑儲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）中準備1至10 μM的工作溶液。對于大多數細胞系，建議終濃度為4至5 μM的細胞活力指標。必須根據經驗確定細胞加載所需指示劑的確切濃度。

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

注意：降低裝載溫度可能會減少指示器的分隔。

4.在無細胞活力指示劑的緩沖液（如果適用，包含陰離子轉運蛋白抑制劑）中洗滌細胞，以去除多余的探針。

注意：立即通過流式細胞儀分析細胞樣品，以測定零時每個細胞的平均熒光強度，無需清洗。

5.在所需的Ex / Em波長下運行實驗。

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