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-mFluor Violet 550测定溶液
3. 在室温下孵育30至60分钟
4. 使用带有525/50 nm滤光片（Pacific Orange通道）的流式细胞仪或带有紫光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-mFluor Violet 550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-mFluor Violet 550（组分A）。

1.5可选：向坏死细胞中加入2 µL 100X碘化丙啶（组分C）。

1.6避光保存，于室温下孵育30至60分钟。

1.7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。

1.8使用带有525/50 nm滤光片的流式细胞仪（Pacific Orange通道）或带有紫色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525/50 nm滤光片（Pacific Orange通道）的流式细胞仪，定量膜联蛋白V-mFluor Violet 550的结合。 将碘化丙啶加入细胞后，使用610/20 nm滤光片（PE-Texas Red 通道）检测细胞活力。 

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悬液，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上。注意：对于贴壁细胞，建议直接在盖玻片上生长细胞。与膜联蛋白V-mFluor Violet 550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-mFluor Violet 550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Violet滤光片组在荧光显微镜下用膜联蛋白V-mFluor Violet 550分析凋亡细胞。当碘化丙锭添加到细胞中时，使用TRITC通道检测细胞活力。质膜上的橙色染色表明膜联蛋白V-mFluor Violet 550与细胞表面的PS结合。

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