Nature Protocols: doi: /nprot Supplementary Figure 1

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1 Supplementary Figure 1 Traditional electronic gating strategy for analysing cell death based on A5-FITC and 7-AAD. a, Flow cytometry analysis showing the traditional two-stage electronic gating strategy used to identify viable cells, A5 + early apoptotic cells and necrotic cells. b, Flow cytometry analysis displaying each type of cells gated according to a has distinctive levels of A5-FITC and 7-AAD staining. Human Jurkat T cells were induced to undergo apoptosis by anti-fas treatment (31.25 ng ml -1, 4 h). Samples were stained with A5-FITC (1 in 2 dilution), 7-AAD (1 μg ml -1 ) and TO-PRO-3 (.5 μm) in 1x A5 Binding Buffer. 3, events (cells and cell fragments) were recorded by the FACSCanto II flow cytometer and resultant data analysed by the FlowJo software. The TO-PRO-3 parameter was not used during data analysis. Nature Protocols: doi:1.138/nprot

2 Supplementary Figure 2 The nucleic acid-binding dye TO-PRO-3 stains viable, early apoptotic and necrotic cells differentially. a, Histogram showing TO-PRO-3 uptake by viable, A5 - early apoptotic, A5 + early apoptotic and necrotic cells. b, Uptake of TO-PRO-3 by cells at different stages of cell death. Data is presented as median fluorescence intensity (MFI) relative to viable cells (n = 3, experimental replicates). Human Jurkat T cells were induced to undergo apoptosis by anti-fas treatment (31.25 ng ml -1, 4 h). Samples were stained with A5-FITC (1 in 2 dilution), 7-AAD (1 μg ml -1 ) and TO-PRO-3 (.5 μm) in 1x A5 Binding Buffer. 3, events (cells and cell fragments) were recorded by the FACSCanto II flow cytometer. Resultant data were analysed by the FlowJo software using the electronic gating strategy as described in Figure 3. Alternative STAGE 1 gating step was used to identify necrotic cells based on the 7-AAD parameter. Error bars represent standard error of the mean. Data are representative of at least two independent experiments. Nature Protocols: doi:1.138/nprot

3 Supplementary Figure 3 Representative images of typical particles in each stage of cell death. ImageStream analysis of particles gated using the strategy as described in Poon et al., Nature 57, (214). Human Jurkat T cells were induced to undergo apoptosis by anti-fas treatment (25 ng ml -1, 4 h). Samples were stained with A5-FITC (1 in 2 dilution), 7-AAD (1 μg ml -1 ) and TO-PRO-3 (.5 μm) in 1x A5 Binding Buffer. Cells and cell fragments were recorded by the ImageStreamX Mark II and resultant data analysed by the IDEAS software. The data shown were previously published in Poon et al., Nature 57, (214). Nature Protocols: doi:1.138/nprot

4 Supplementary Figure 4 Electronic gating strategy for analysing cell death based only on TO-PRO-3. Flow cytometry analysis showing a one-stage electronic gating strategy used to identify viable cells, early apoptotic cells (A5 - and A5 + ), necrotic cells, as well as apoptotic bodies and debris. Human Jurkat T cells were induced to undergo apoptosis by anti-fas treatment (31.25 ng ml -1, 4 h). Samples were stained with A5-FITC (1 in 2 dilution), 7-AAD (1 μg ml -1 ) and TO-PRO-3 (.5 μm) in 1x A5 Binding Buffer. 3, events (cells and cell fragments) were recorded by the FACSCanto II flow cytometer and resultant data analysed by the FlowJo software. The A5-FITC and 7-AAD parameter was not used during data analysis. Nature Protocols: doi:1.138/nprot

5 Supplementary methods 1 " Human THP-1 monocytic cells (a) were induced to undergo apoptosis by ultraviolet C irradiation (15 mj cm -2 ). THP-1 cells were analysed by flow cytometry 4 h post ultraviolet C irradiation treatment. Primary human peripheral blood monocytes (b) were induced to undergo apoptosis by serum-starvation for 4 h prior to flow cytometry analysis. Primary mouse thymocytes (c) were induced to undergo apoptosis by dexamethasone treatment (5 μm) for 5 h prior to flow cytometry analysis. These non-adherent cells were prepared as described in Procedure. Isolation of primary human peripheral blood monocytes and primary mouse thymocytes were performed according to Atkin-Smith et al. 2 and Poon et al. 1, respectively. Adherent cells including human A431 squamous carcinoma cells (d), human umbilical vein endothelial cells (e), and mouse embryonic fibroblasts (f), were induced to undergo apoptosis by ultraviolet C irradiation (15 mj cm -2 ). These adherent cells were analysed by flow cytometry 4 h post ultraviolet C irradiation treatment. For human A431 squamous carcinoma cells and human umbilical vein endothelial cells, only cells that have already detached and are in the culture supernatant were analysed. For mouse embryonic fibroblasts, cells in the culture supernatant and cells dissociated from the cell culture surface via trypsin/edta treatment were analysed together. See Introduction for details regarding the preparation of adherent cells.# Nature Protocols: doi:1.138/nprot

6 a" Human THP-1 monocytic cells + ultraviolet C irradiation# b" Primary human peripheral blood monocytes + serum-starvation# 25K K 1 5 2K 2K K 15K K 1K 5K 1 2 5K 1 2 5K 1K 15K 2K 25K K 1K 15K 2K 25K c" d" Primary mouse thymocytes + dexamethasone# Human A431 squamous carcinoma + ultraviolet C irradiation# 25K K K K 4 5K K 1K 15K 2K 25K K K K 1K 5K 5K 1K 15K 2K 25K Nature Protocols: doi:1.138/nprot

7 e" Human umbilical vein endothelial cells + ultraviolet C irradiation# f" Mouse embryonic fibroblasts + ultraviolet C irradiation# K K K K K K 1K 15K 2K 25K K K K 1K 5K 5K 1K 15K 2K 25K Supplementary Data 1. Identification of cells at different stages of cell death and cell fragments generated from various cell types. Flow cytometry analysis displaying each type of cells and cell fragments gated according to Figure 3 and Table 2 has distinctive levels of A5-FITC and TO-PRO-3 staining, as well as FSC and SSC properties. Top panels are representative flow cytometry plots of FSC versus SSC and A5-FITC versus TO-PRO-3 generated from raw data. In the bottom panel, cells at different stages of cell death and cell fragment type are plotted based on FSC versus SSC and A5-FITC versus TO-PRO-3. Non-adherent cells include human THP-1 monocytic cells (a), primary human peripheral blood monocytes (b) and primary mouse thymocytes (c). Adherent cells include human A431 squamous carcinoma cells (d), human umbilical vein endothelial cells (e), and mouse embryonic fibroblasts (f). All cell lines used in this research were regularly checked to ensure they are not infected with mycoplasma. # Nature Protocols: doi:1.138/nprot

8 Supplementary Reference 1 Poon, I. K. et al. Unexpected link between an antibiotic, pannexin channels and apoptosis. Nature 57, (214). 2 Atkin-Smith, G. K. et al. A novel mechanism of generating extracellular vesicles during apoptosis via a beads-on-a-string membrane structure. Nature Communications 6, 7439 (215). Nature Protocols: doi:1.138/nprot