适用仪器

样品实验方案

简要概述

1. 用5×10⁵至1×10⁶细胞/ mL的密度制备含测试化合物的细胞
2. 将0.5 µL细胞溶液中加入1 µL 500X TF5-VAD-FMK
3. 将细胞在37°C，5％CO₂培养箱中孵育1-4小时
4. 沉淀细胞并将细胞重悬于0.5 mL检测缓冲液或生长培养基中
5. 使用带有660/20 nm滤光片的流式细胞仪分析细胞（APC通道）

实验步骤

1.对于每个样品，在0.5mL温热培养基或自备缓冲液中以5×10⁵至1×10⁶cell/ mL的密度制备细胞。注意：应单独评估每种细胞系，以确定诱导凋亡的最佳细胞密度。

2.用测试化合物处理细胞一段时间，以诱导细胞凋亡，并建立阳性和阴性对照。

3.向处理过的细胞中加入1µL 500X TF5-VAD-FMK（组分A）。

4.将细胞在37°C，5％CO₂培养箱中孵育1-4小时。注意：对于贴壁细胞，用0.5 mM EDTA轻轻提起细胞以保持细胞完整，并在与TF5-VAD-FMK孵育之前用含血清的培养基洗涤细胞一次。合适的孵育时间取决于所用的单个细胞类型和细胞浓度。优化每个实验的孵育时间。

5.洗涤并旋转细胞两次。将细胞重悬于0.5 mL分析缓冲液（组分B）或生长培养基中。注意：TF5-VAD-FMK是荧光的；因此，重要的是洗净任何未结合的试剂以除去背景。

6.可选：用DNA染色剂标记细胞（例如绿色DCS标记死细胞，可以用530/30 nm滤光片（FITC通道）检测到。

7.可选：修复细胞。

8.使用带有660/20 nm滤光片（APC通道）的流式细胞仪检测荧光强度。

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