活性氧 Cell Meter 荧光法线粒体超氧化物检测试剂盒

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流式细胞仪
Ex:	488 nm
Em：	575/26  nm
通道:	PE 通道

样品实验方案

简要概述

1. 准备细胞密度为0.5-1×10 ⁶细胞/ mL
2. 用测试化合物处理细胞以诱导超氧化物
3. 将1 µL 500X MitoROS 580加到0.5 mL细胞悬液中
4. 将细胞在37°C染色1小时
5. 使用带有FL2通道的流式细胞仪检测荧光强度

 

溶液配制

储备溶液配制

1. MitoROS 580储备溶液（500X）：将100 µL DMSO（组分C）添加到MitoROS 580（组分A）小瓶中，并充分混合以制成500X MitoROS 580储备溶液。避光。注意：请密闭存放。

 

实验步骤

1. 对于每个样品，在自备的0.5 mL生长培养基或缓冲液中制备细胞，密度为5×10 ⁵至1×10 ⁶个细胞/ mL。注意：应单独评估每种细胞系，以确定超氧化物诱导的细胞密度。
   
   
2. 在分析缓冲液（组分B）或自备的缓冲液（例如PBS）中用25 µL 20X测试化合物处理细胞以诱导超氧化物。对于对照细胞（未处理的细胞），添加相应量的化合物缓冲液。
   
   
3. 将细胞在37°C孵育至少30分钟。注意：将 Jurkat细胞在37°C下用50 µM抗霉素A（AMA）处理30分钟以诱导超氧化物。有关详细信息，请参见图1。
   
   
4. 将0.5 µL细胞悬浮液中加入1 µL 500X MitoROS 580储备液。
   
   
5. 在37°C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MitoROS 580孵育之前用含血清的培养基洗涤一次。适当的孵育时间取决于单个细胞的类型和测试使用的化合物。
6. 使用带有FL2通道的流式细胞仪（Ex / Em = 488/590 nm）检测荧光强度。

 图示

 

 

图1.使用Cell Meter 荧光细胞内超氧化物检测试剂盒检测Jurkat细胞中的超氧化物。AMA处理（红色）：将细胞在37°C下用50 µM抗霉素A（AMA）处理30分钟，然后与MitoROS 580孵育1小时。对照（蓝色）：在未经AMA处理的情况下，将细胞与MitoROS 580在37°C孵育1小时。使用流式细胞仪（BD FACSCalibur）在FL2通道检测上荧光信号。

 

 

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