钙黄绿素橙钠盐是美国AAT Bioquest生产的用于细胞活力测定的试剂,Calcein AM 是流行的荧光探针之一,用于标记和监测活细胞的细胞功能。百萤生物是AAT Bioquest 的中国代理商,为您提供优质的钙黄绿素橙钠盐。
光谱:
实验方案;
使用细胞活力指标AM酯类
AM酯是非极性酯,其易于穿过活细胞膜,并且通过活细胞内的细胞酯酶快速水解。 AM酯广泛用于非侵入性地将各种极性荧光探针加载到活细胞中。 但是,使用AM酯时必须小心,因为它们易于水解,特别是在溶液中。 它们应在使用前重新配制成高质量的无水二甲基亚砜(DMSO)。 DMSO储备溶液可以在20℃下干燥储存并避光。 在这些条件下,AM酯应稳定数月。
以下是我们推荐的将AM酯加载到活细胞中的方案。 该说明书仅提供指南,实际应根据您的具体需求进行修改。
a)在高质量无水DMSO中制备2至5mM AM酯的储备溶液。非离子洗涤剂Pluronic®F-127有时用于增加AM酯的水溶性。
注意:在稀释到上样缓冲液中之前,可将等体积的20%Pluronic®F-127溶液添加到细胞活力指示剂的DMSO储备溶液中。终的Pluronic®F-127浓度约为0.02%。
注意:不建议在Pluronic®F-127存在下长期储存AM酯。
b)在实验当天,要么将细胞活力的固体指示剂溶解在DMSO中,要么将等分的指示剂储备溶液解冻至室温。在您选择的缓冲液(如Hanks和Hepes缓冲液)中制备1至10μM的工作溶液。对于大多数细胞系,建议终浓度范围为4至5μM的细胞活力指标。细胞加载所需指标的确切浓度必须根据经验确定。
c)如果您的细胞含有有机阴离子转运蛋白,可以在细胞培养基中加入丙磺舒(1-2.5 mM)或磺吡酮(0.1-0.25 mM),以减少脱酯化指标的泄漏。在室温或37℃下将细胞与AM酯孵育20分钟至1小时。
d)在不含细胞活力指示剂的缓冲液(含有阴离子转运蛋白抑制剂,如果适用)中洗涤细胞以除去过量的探针。
注意:通过流式细胞术立即分析细胞样品,以确定在零时间没有洗涤的每个细胞的平均荧光。
e)在所需的Ex / Em波长下进行实验 。
图1所示。与钙黄绿素的形象活海拉细胞染色橙色™钠盐(# 22008)和钙黄绿素二乙酸橙™(# 22009)。钙黄绿素橙色™钠盐不能渗透完整活细胞
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