?鈣黃綠素橙鈉鹽是美國AAT Bioquest生產(chǎn)的用于細(xì)胞活力測(cè)定的試劑，Calcein AM 是流行的熒光探針之一，用于標(biāo)記和監(jiān)測(cè)活細(xì)胞的細(xì)胞功能。百螢生物是AAT Bioquest?的中國代理商，為您提供優(yōu)質(zhì)的鈣黃綠素橙鈉鹽。
光譜：
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實(shí)驗(yàn)方案;
使用細(xì)胞活力指標(biāo)AM酯類
? ? ? ? AM酯是非極性酯，其易于穿過活細(xì)胞膜，并且通過活細(xì)胞內(nèi)的細(xì)胞酯酶快速水解。 AM酯廣泛用于非侵入性地將各種極性熒光探針加載到活細(xì)胞中。 但是，使用AM酯時(shí)必須小心，因?yàn)樗鼈円子谒猓貏e是在溶液中。 它們應(yīng)在使用前重新配制成高質(zhì)量的無水二甲基亞砜（DMSO）。 DMSO儲(chǔ)備溶液可以在20℃下干燥儲(chǔ)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。 該說明書僅提供指南，實(shí)際應(yīng)根據(jù)您的具體需求進(jìn)行修改。?
a）在高質(zhì)量無水DMSO中制備2至5mM AM酯的儲(chǔ)備溶液。非離子洗滌劑Pluronic?F-127有時(shí)用于增加AM酯的水溶性。
注意：在稀釋到上樣緩沖液中之前，可將等體積的20％Pluronic?F-127溶液添加到細(xì)胞活力指示劑的DMSO儲(chǔ)備溶液中。終的Pluronic?F-127濃度約為0.02％。
注意：不建議在Pluronic?F-127存在下長期儲(chǔ)存AM酯。
b）在實(shí)驗(yàn)當(dāng)天，要么將細(xì)胞活力的固體指示劑溶解在DMSO中，要么將等分的指示劑儲(chǔ)備溶液解凍至室溫。在您選擇的緩沖液（如Hanks和Hepes緩沖液）中制備1至10μM的工作溶液。對(duì)于大多數(shù)細(xì)胞系，建議終濃度范圍為4至5μM的細(xì)胞活力指標(biāo)。細(xì)胞加載所需指標(biāo)的確切濃度必須根據(jù)經(jīng)驗(yàn)確定。
c）如果您的細(xì)胞含有有機(jī)陰離子轉(zhuǎn)運(yùn)蛋白，可以在細(xì)胞培養(yǎng)基中加入丙磺舒（1-2.5 mM）或磺吡酮（0.1-0.25 mM），以減少脫酯化指標(biāo)的泄漏。在室溫或37℃下將細(xì)胞與AM酯孵育20分鐘至1小時(shí)。
d）在不含細(xì)胞活力指示劑的緩沖液（含有陰離子轉(zhuǎn)運(yùn)蛋白抑制劑，如果適用）中洗滌細(xì)胞以除去過量的探針。
注意：通過流式細(xì)胞術(shù)立即分析細(xì)胞樣品，以確定在零時(shí)間沒有洗滌的每個(gè)細(xì)胞的平均熒光。
e）在所需的Ex / Em波長下進(jìn)行實(shí)驗(yàn) 。
圖1所示。與鈣黃綠素的形象活海拉細(xì)胞染色橙色?鈉鹽(# 22008)和鈣黃綠素二乙酸橙?(# 22009)。鈣黃綠素橙色?鈉鹽不能滲透完整活細(xì)胞
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