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 500分析溶液
3. 在室温下孵育30-60分钟
4. 使用带有525/40 nm滤光片（AmCyan通道）的流式细胞仪或带有紫色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 500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在细胞中加入2 µL Annexin V-mFluor Violet 500（组分A）。

1.5可选：向坏死的细胞中加入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525/40 nm滤光片的流式细胞仪（AmCyan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525/40 nm滤光片的流式细胞仪（AmCyan通道）定量膜联蛋白V-mFluor Violet 500结合。 将碘化丙啶加入细胞后，使用610/20 nm滤光片（PE-德克萨斯红通道）测量细胞活力。 注意：对贴壁细胞的膜联蛋白V结合流式细胞术分析未经过常规测试，因为在细胞分离或收获过程中可能会发生特定的膜损伤。 

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。与膜联蛋白V-mFluor Violet 500一起孵育后，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 500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使用紫罗兰滤光片组在荧光显微镜下用膜联蛋白V-mFluor Violet 500分析凋亡细胞。当碘化丙锭添加到细胞中时，使用TRITC通道测量细胞活力。质膜上的绿色染色表明膜联蛋白V-mFluor Violet 500与细胞表面的PS结合。

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