适用仪器

样品实验方案

简要概述

1. 用测试化合物制备细胞（200 µL /样品）
2. 添加膜联蛋白V-iFluor 647测定溶液
3. 在室温下孵育30-60分钟
4. 使用带有660/20 nm滤光片（APC通道）的流式细胞仪或带有Cy5滤光片组的荧光显微镜分析细胞

实验步骤

1.用膜联蛋白V-iFluor 647制备和孵育细胞：

1.1用测试化合物处理细胞一段时间（对于用星形孢菌素处理过的Jurkat细胞，需要4-6小时）以诱导细胞凋亡。

1.2离心细胞以获得1-5×105细胞/管。

1.3将细胞重悬于200 µL分析缓冲液（组分B）中。

1.4向细胞中加入2 µL Annexin V-iFluor 647（组分A）。

1.5避光保存，于室温下孵育30至60分钟。

1.6在使用流式细胞仪或荧光显微镜分析细胞之前，添加300 µL分析缓冲液（组分B）以增加体积。

1.7使用带有660/20 nm滤光片的流式细胞仪（APC通道）或带有Cy5滤光片组的荧光显微镜检测荧光强度。

2.通过使用流式细胞仪进行分析：

2.1使用带有660/20 nm滤光片（APC通道）的流式细胞仪定量膜联蛋白V-iFluor 647的结合物。

3.通过使用荧光显微镜进行分析：

3.1孵育后用移液管吸移细胞，用测定缓冲液冲洗1-2次，然后用测定缓冲液重悬细胞。

3.2将细胞添加到载玻片盖玻片的载玻片上。 注意：对于贴壁细胞，建议直接在盖玻片上生长细胞。 与Annexin V-iFluor 647一起孵育后，用测定缓冲液冲洗1-2次，然后将测定缓冲液加回到盖玻片上。 将玻片上的盖玻片倒置并可视化细胞。 与膜联蛋白V-iFluor 647孵育后，还可以将细胞固定在2％甲醛中，并在显微镜下观察。

3.3使用Cy5滤光片组，在荧光显微镜下用Annexin V-iFluor 647分析凋亡细胞。当碘化丙锭添加到细胞中时，通过使用TRITC通道测量细胞活力。质膜上的橙色染色表明膜联蛋白V-iFluor 647与细胞表面的PS结合。

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