样品分析方案
概述
用测试化合物(200μL/样品)制备细胞
添加膜联蛋白V缀合物测定溶液
室温孵育30-60分钟
用流式细胞仪或荧光显微镜分析
操作步骤
1.用膜联蛋白V缀合物制备和孵育细胞:
1.1制备膜联蛋白V结合测定缓冲液:10mM HEPES,140mM NaCl和2.5mM CaCl 2,pH 7.4。
1.2用测试化合物处理细胞一段所需的时间(用星形孢菌素处理的Jurkat细胞4-6小时)以诱导细胞凋亡。
1.3离心细胞,得到1-5×105个细胞/管。
1.4将细胞重悬于200μL膜联蛋白V结合测定缓冲液中(来自步骤1.1)。
1.5在细胞中加入2μL膜联蛋白V结合物。
可选:在细胞内加入死细胞染色剂如碘化丙锭,用于坏死细胞。
1.6在室温下孵育30至60分钟,避光。
1.7在用流式细胞仪或荧光显微镜分析细胞之前,加入300μL膜联蛋白V结合测定缓冲液(来自步骤1.1)以增加体积(参见下面的步骤1.8)。
1.8使用流式细胞仪或荧光显微镜监测荧光强度(参见下面的步骤2或3)。
2.使用流式细胞仪分析:
通过使用具有适当过滤器的流式细胞仪来量化膜联蛋白V缀合物。
注意:粘附细胞上的膜联蛋白V结合流式细胞术分析未经常检测,因为在细胞分离或收获期间可能发生特定的膜损伤。 然而,Casiola-Rosen等人先前报道了利用膜联蛋白V对贴壁细胞类型进行流式细胞术的方法。 和van Engelend等人(见参考文献1和2)。
3.使用荧光显微镜分析:
3.1移取步骤1.6的细胞悬液,用膜联蛋白V结合测定缓冲液(来自步骤1.1)冲洗1-2次,然后用膜联蛋白V结合测定缓冲液(来自步骤1.1)重悬细胞。 将细胞添加到盖有玻璃盖玻片的载玻片上。
注意:对于粘附细胞,建议直接在盖玻片上生长细胞。 与膜联蛋白V缀合物孵育(步骤1.6)后,用膜联蛋白V结合测定缓冲液(来自步骤1.1)冲洗1-2次,并将膜联蛋白V结合测定缓冲液(来自步骤1.1)加回到盖玻片中。 在玻璃载玻片上翻转盖玻片并观察细胞。 在与膜联蛋白V缀合物孵育后,细胞也可以在2%甲醛中固定,并在显微镜下观察。
3.2在荧光显微镜下用适当的滤光片分析膜联蛋白V缀合物的凋亡细胞。
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