适用仪器

样品实验方案

简要概述

1. 在生长培养基中准备细胞
2. 加入Amplite ROS Red工作溶液（对于96孔板为100 µL /孔，对于384孔板为25 µL /孔）
3. 在37°C下孵育细胞1小时
4. 用测试化合物处理细胞以诱导ROS
5. 在Ex / Em = 520/605 nm（截止= 590 nm）或安装Ex / Em = 520/605 nm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

储备溶液配制

1. Amplite ROS Red储备液（500X）：将40 µL DMSO（组分C）添加到Amplite ROS Red（组分A）的小瓶中，并充分混合以制成500X Amplite ROS Red储备液。 避光。 注意：20 µL 500X Amplite ROS Red储备液足以用于1个板。 注意：如果将试管紧密密封并避免光照，可以将未使用的部分等分并在<-20°C下保存超过一个月。 避免重复冻融循环。

工作溶液配制

将20 µL 500X Amplite ROS Red储备溶液添加到10 mL的测定缓冲液（组分B）中，并充分混合以制成Amplite ROS Red工作溶液。 注意：此Amplite ROS Red工作溶液在室温下至少可稳定2小时。

实验步骤

1.将100 µL /孔（96孔板）或25 µL /孔（384孔板）的Amplite ROS Red工作溶液添加到细胞板中。

2.将细胞在5％CO₂、37°C的培养箱中孵育一小时。

3.在所需的缓冲液（例如PBS或HHBS）中，用20µL 11X测试化合物（96孔板）或10 µL 6X测试化合物（384孔板）处理细胞。 对于对照孔（未处理的细胞），添加相应量的化合物缓冲液。

4.要诱导ROS，请在室温下或在5％CO₂、37°C的培养箱中孵育细胞板至少15分钟（对于用1 mM H₂O₂处理的Hela细胞为30分钟）。

5.在Ex / Em = 520/605 nm（截止= 590 nm）处使用荧光酶标仪（底部读取模式）检测荧光的增加，或在Ex / Em = 520/605 nm滤光片组（Texas Red）下使用荧光显微镜观察细胞）。

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