分析方案
概述
准备细胞
添加染料工作溶液
在37°C孵育30分钟至2小时
在荧光显微镜下Ex / Em = 405 / 480nm(紫外滤光片组)处进行分析
操作方法
1.准备溶酶体染色溶液:
1.1解冻 Lysolite VLG26(组分A)至室温。
1.2通过将20μLLysolite VLG26(组分A)稀释到10mL活细胞染色缓冲液(组分B)中制备染料工作溶液。
注1:对于一个96孔板,20μLLysolite VLG26(组分A)就足够了。 将未使用的Lysolite VLG26(组分A)等分并储存在<-20℃。 避光,避免反复冻融循环。
注2:荧光溶酶体指示剂的浓度根据具体应用而变化。 可以根据特定细胞类型和细胞或组织对探针的渗透性来修改染色条件。
2.准备和染色细胞:
2.1对于粘附细胞:在96孔黑色壁/透明底板(100μL/孔/ 96孔板)中或在装有适当培养基的培养皿内的盖玻片上培养细胞。当细胞达到所需的汇合时,加入等体积(如100μL/孔/ 96孔板)的染料加工溶液(来自步骤1.2)。将细胞在37℃,5%CO2培养箱中孵育30分钟至2小时。使用配有Violet滤光片组(Ex / Em = 405 / 480nm)的荧光显微镜观察细胞。
注意:如果细胞看起来没有充分染色,建议增加标记浓度或孵育时间以使染料积累。
2.2对于悬浮细胞:以1,000rpm离心细胞5分钟以获得细胞沉淀并吸出上清液。在预热的生长培养基中轻轻重悬细胞沉淀,然后加入等体积的染料加工溶液(来自步骤1.2)。将细胞在37℃,5%CO2培养箱中孵育30分钟至2小时。使用配有Violet滤光片组(Ex / Em = 405 / 480nm)的荧光显微镜观察细胞。
注1:如果细胞看起来没有充分染色,建议增加标记浓度或培养时间以使染料积累。
注2:悬浮细胞可以附着在用BD Cell-Tak®(BD Biosciences)处理过的盖玻片上,并作为贴壁细胞染色(见步骤2.1)。
图1.使用Cell Navigator 溶酶体染色试剂盒染色的U2OS细胞图像使用Costar黑色96孔板进行405 nm激发的绿色荧光
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