样品实验方案
简要概述
1.用测试化合物制备细胞(100 µL /孔/ 96孔板或25 µL /孔/ 384孔板)
2.加入等量的Apopxin™Violet 450工作溶液
3.在室温下孵育1小时
4.观察Ex / Em = 405/450 nm(截止= 420 nm)的荧光强度(底部读取模式)或使用装有紫罗兰色滤光片的荧光显微镜
溶液配制
工作溶液配制
将10μL100X Apopxin™紫罗兰色450(组分A)添加到1 mL的测定缓冲液(组分B)中,并充分混合以制成Apopxin™紫罗兰色450工作溶液。
操作步骤
1.通过向PBS或所需的缓冲液中加入10X /孔(96孔板)或2.5 µL /孔(384孔板)的10X测试化合物储备溶液来处理细胞。对于空白孔(不含细胞的培养基),添加相同量的化合物缓冲液。
2.将细胞板在5%CO2、37°C的培养箱中孵育所需的时间(对于用星形孢菌素处理的Jurkat细胞需要4-6小时)以诱导凋亡。注意:由于在405 nm激发时,从380至490 nm的宽发射光谱,某些化合物(例如喜树碱)可能会产生假阳性反应。
3.在每个孔中添加100 µL /孔(96孔板)或25 µL /孔(384孔板)的Apopxin™Violet 450工作溶液。
4.在避光条件下,于室温下孵育细胞板至少1小时。
5.以800 rpm的速度离心细胞板(特别是非粘附细胞)2分钟(制动)。
6.使用荧光酶标仪(底部读取模式)在Ex / Em = 405/450 nm(截止= 420 nm)或使用带有紫色滤光片的荧光显微镜对图像池进行荧光强度监测。
图1.用Apopxin™Violet 450偶联物染色的HeLa细胞的荧光图像。 Jurkat细胞在37℃下不使用(左)或用1μM星形孢菌素(右)处理4小时。 使用带有紫色滤光片组的显微镜(激发= 405nm)测量荧光强度。
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