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的最佳细胞密度。
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
图1.使用Cell Meter 荧光法细胞内总ROS活性测定试剂盒在TBHP处理后检测Jurkat细胞中的细胞内ROS。 将细胞与Amplite ROS Green在37°C孵育1小时。 然后将细胞在不使用(蓝色)或(红色)100 µM TBHP的情况下在37°C下处理30分钟。 使用流式细胞仪(BD FACSCalibur)在FL1通道处检测荧光信号。 |
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