Published in

Nature Research, Nature Protocols, 4(11), p. 655-663, 2016

DOI: 10.1038/nprot.2016.028

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Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry

This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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Abstract

The use of annexin A5 (A5 )and propidium iodide of 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, that is 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 utilises a combination of A5 and TO-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. This protocol requires 1-5 h for sample preparation (including induction of cell death), 20 min for staining and 5 min for data analysis.