适用仪器

样品实验方案

简要概述

1. 准备细胞
2. 添加测试化合物
3. 添加MitoTell Red工作溶液（100 µL /孔/ 96孔板或25 µL /孔/ 384孔板）
4. 将板在5％CO₂、37°C的培养箱中孵育30分钟
5. 添加测定缓冲液B（50 µL /孔/ 96孔板或12.5 µL /孔/ 384孔板）
6. 在Ex / Em = 610/650 nm（截止= 630 nm）或使用Cy5滤光片的荧光显微镜下检测荧光增加（底部读取模式）

溶液配制

将50 µL的200X MitoTell Red（组分A）添加到10 mL的测定缓冲液A（组分B）中，并充分混合以制成MitoTell Red工作溶液，避光。

实验步骤

1.用测试化合物处理细胞一段时间，以诱导细胞凋亡，并建立平行对照实验。注意：我们用20 µM CCCP处理HeLa细胞15分钟，以改变线粒体膜电位。CCCP或FCCP可以与MitoTell Red同时添加。为了获得最佳结果，可能需要为每个单独的细胞系滴定CCCP或FCCP。

2.将100 µL /孔/ 96孔板或25 µL /孔/ 384孔板的MitoTell Red工作溶液加入细胞板。

3.在避光的条件下，将板在37ºC下孵育15-30分钟。注意：适当的孵育时间取决于所用的单个细胞类型和细胞浓度。优化每个实验的孵育时间。

4.将50 µL /孔/ 96孔板或12.5 µL /孔/ 384孔板的测定缓冲液B（组分C）添加到细胞板中。注意：加载后请勿洗涤细胞。对于非贴壁细胞，建议在加入测定缓冲液B（组分C）后，以800 rpm离心细胞板2分钟，然后制动。

5.加入测定缓冲液B（组分C）后10到30分钟，用荧光酶标仪（Ext / Em = 610/650 nm（截止= 630 nm））检测荧光强度，或在荧光下用带Cy5滤镜的显微镜观察荧光信号。

参考文献

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