样品实验方案
简要概述
1.使用96孔板的密度为5×104至2×105细胞/ 100 µL /孔的待测化合物制备细胞
2.加入等体积的ApoSight Caspase 3/7底物工作溶液
3.在5%CO2培养箱中于37°C孵育60分钟
4.用HHBS洗涤细胞1-2次
5.使用带有530/30 nm滤波片(FITC通道)的流式细胞仪或带有FITC滤波片组的荧光显微镜分析细胞
溶液配制
1.储备溶液配制
所有未使用的储备溶液应分为一次性使用的等分试样,并在制备后储存在-20°C下。 避免重复冻融循环。
ApoSight Green Caspase 3/7底物储备液(200X)
在ApoSight 绿色胱天蛋白酶3/7底物瓶中加入50 µL DMSO,制成200X ApoSight 绿色胱天蛋白酶3/7底物原液。
注意,一次性使用等分试样,以避免重复的冻融循环。
2.工作溶液配置
ApoSight Green Caspase 3/7底物工作溶液
通过将5 µL 200X ApoSight Green Caspase 3/7底物储备溶液与1 mL分析缓冲液(组分B)混合,制备ApoSight Green Caspase 3/7底物工作溶液。
注意,100 µL ApoSight Green Caspase 3/7底物工作溶液足以进行96孔板的10次检测
注意,实验前准备足够的ApoSight Green Caspase 3/7底物工作溶液,并立即使用。
样品示例及操作
悬浮培养中诱导细胞凋亡的实例
1.用2μg/ mL喜树碱处理Jurkat细胞3小时
2.用1μM星形孢菌素处理Jurkat细胞3-4小时
3.用4μg/ mL喜树碱处理HL-60细胞4小时
4.用1μM星形孢菌素处理HL-60细胞4小时。
注意,应对每个细胞系进行单独评估,以确定诱导凋亡的最佳细胞密度。
荧光显微镜的样品方案
1.用5×104至2×105个细胞/ 100 µL /孔/ 96孔板的密度制备含待测化合物的细胞。
2.向细胞中加入等体积的Caspase 3/7底物工作溶液(100 µL /孔/ 96孔板)。
3.在5%CO2培养箱中于37°C孵育60分钟。
4.用HHBS或您选择的缓冲液洗涤细胞1-2次。
5.使用FITC滤波片组的荧光显微镜成像。
图示
图1.使用ApoSight 绿色胱天蛋白酶3/7底物检测Jurkat细胞中胱天蛋白酶3/7活性。将Jurkat细胞(200,000个细胞/孔/ 96孔板)用1μM星形孢菌素或DMSO处理4小时。将细胞与Caspase 3/7底物工作溶液在37°C下孵育1小时。使用FITC滤波片组,用荧光显微镜拍摄图像。
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