适用仪器

样品实验方案

简要概述

1.准备密度为0.5-1×10⁶细胞/ mL的细胞
2.在0.5 mL细胞悬液中加入1 µL 500X Amplite ROS Green
3.在37℃下将细胞染色1小时
4.处理细胞以诱导ROS
5.使用带有FL1通道的流式细胞仪分析细胞（Ex / Em = 490/520 nm）

溶液配制

1.储备溶液配制

        除非另有说明，否则所有未使用的储备溶液应分成一次性等分试样，并在制备后储存在-20°C。 避免反复冻融循环。

Amplite ROS绿色储备溶液（500X）：将100 µL DMSO（组分C）添加到Amplite ROS Green（组分A）的小瓶中，并充分混合以制成500X Amplite ROS Green储备液。 避光。 注意：要存放，请密封管。有关细胞样品制备的指南，请点击查看。

操作步骤

1.对于每个样品，以0.5×10⁵至1×10⁶细胞/ mL的密度在0.5 mL测定缓冲液（组分B）或自备缓冲液中制备细胞。注意：应单独评估每个细胞系，以确定诱导ROS的细胞密度。

2.将1 µL 500X Amplite ROS Green储备溶液加入0.5 mL细胞悬液中。

3.在37ºC下孵育1小时。注意：对于贴壁细胞，用0.5 mM EDTA轻轻提起细胞以保持细胞完整，并在与Amplite ROS Green孵育之前用含血清的培养基洗涤细胞一次。合适的孵育时间取决于所用的单个细胞类型和测试化合物，优化每个实验的孵育时间。

4.通过在所需的缓冲液（例如PBS或HBSS）中添加50 µL 11X测试化合物来处理细胞。对于对照孔（未处的细胞），添加相应量的缓冲液。

5.将细胞在37ºC下孵育，以诱导ROS（避光）。注意：我们在37℃下用100 µM TBHP（氢过氧化叔丁基）处理Jurkat细胞30分钟，以诱导ROS。有关详细信息，请参见图1。

6.使用具有FL1通道（Ex / Em = 490/520 nm）的流式细胞仪检测荧光强度。

图示

参考文献

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