Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry
Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry
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DOI：
10.1038/nprot.2016.028
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发表时间：
2016-04-01
期刊：
影响因子：
14.8
通讯作者：
Poon, Ivan K. H.
中科院分区：
文献类型：
--
作者：
Jiang, Lanzhou;Tixeira, Rochelle;Poon, Ivan K. H.
使用膜联蛋白A5（A5）和碘化丙啶或7-氨基放线菌素D（PI/7-AAD）染色通过流式细胞术测量细胞死亡被大多数研究者认为是金标准。然而，这种广泛使用的方法通常假设样品中只有三种类型的颗粒：活细胞，凋亡细胞和坏死细胞。为了更详细地研究细胞死亡的进展，特别是凋亡细胞如何经历碎片化以产生称为凋亡小体的膜结合囊泡，我们建立了一种基于流式细胞术的方案来准确、快速地测量细胞死亡过程。该方案使用A5和T0-PRO-3（一种可商购的核酸结合染料，对早期凋亡和坏死细胞进行差异染色）的组合，以及逻辑七阶段分析方法来区分样品中的六种类型的颗粒，包括凋亡小体和处于细胞死亡三个不同阶段的细胞。该方案需要1-5 h用于样品制备（包括诱导细胞死亡），20 min用于染色，5 min用于数据分析。
The use of annexin A5 (A5) and either propidium iodide or 7-aminoactinomycin D (PI/7-AAD) stains to measure cell death by flow cytometry has been considered the gold standard by most investigators. However, this widely used method often makes the assumption that there are only three types of particles in a sample: viable, apoptotic and necrotic cells. To study the progression of cell death in greater detail, in particular how apoptotic cells undergo fragmentation to generate membrane-bound vesicles known as apoptotic bodies, we established a flow cytometry-based protocol to accurately and rapidly measure the cell death process. This protocol uses a combination of A5 and T0-PRO-3 (a commercially available nucleic acid-binding dye that stains early apoptotic and necrotic cells differentially), and a logical seven-stage analytical approach to distinguish six types of particles in a sample, including apoptotic bodies and cells at three different stages of cell death. The protocol requires 1-5 h for sample preparation (including induction of cell death), 20 min for staining and 5 min for data analysis.