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

Abstract

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 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. The protocol requires 1-5 h for sample preparation (including induction of cell death), 20 min for staining and 5 min for data analysis.

Isolation of cell type-specific apoptotic bodies by fluorescence-activated cell sorting

“…1d). Consistent with previous studies, ApoBDs isolated via FACS exhibited intermediate TO-PRO-3 and A5 staining11011 (Fig. 1e, Supplementary Fig.…”

“…Recently, we established a cell staining and flow cytometry approach that can identify ApoBDs from viable, apoptotic and necrotic cells based on relative size and granularity, in addition to differential TO-PRO-3 and annexin A5 (A5) staining11011. By further adapting this analytical method, we can use FACS to isolate ApoBDs from a cell culture sample.…”

Phosphatidylserine externalization, “necroptotic bodies” release, and phagocytosis during necroptosis

“…These findings suggest that PS externalization, and its detection by annexin V (A5), cannot be used as a distinguishing marker between apoptosis and regulated necrosis [21–24]. …”

A Maleimide‐functionalized Tetraphenylethene for Measuring and Imaging Unfolded Proteins in Cells

“…First, unfolded proteins were induced in cells by different stressors in the same way as conducting cell imaging. Second, cells were stained by TPE‐NMI and then TO‐PRO‐3, a cell‐impermeant nucleic acid stain to differentiate live/dead cells . Finally, TPE‐NMI signal from live single cells was collected and analyzed by flow cytometry, and the gating strategies are shown in Figure S12.…”