Fig 1. Detection of A5 single-positive cells during necroptosis.

(A–C) L929 cells were stimulated for either (i) apoptosis (TS), necroptosis (TSZ), or (ii) left untreated (Con). Where indicated, RIPK1 (Nec-1s) and RIPK3 (gsk872) inhibitors were added to the cells 30 minutes prior to TSZ stimulation. (A) 10⁶ cells were harvested 3 hours after stimulation of cell death, and cell death key factors pMLKL and CC3 were detected using western blot. (B) Cell viability was measured at different time points after stimulation of cell death using A5/PI staining and then analyzed by flow cytometry (mean ± SD). (C) Example single A5-FITC-positive necroptotic cells, imaged using live microscopy. (D–F) U937 cells were stimulated for either (i) apoptosis (TS), necroptosis (TSZ) or (ii) left untreated (Con). Where indicated, RIPK1 (Nec-1s), RIPK3 (gsk872) and pMLKL (NSA) inhibitors were added to the cells 30 minutes prior to TSZ stimulation. (D) Cell viability was measured at different time points after stimulation of cell death using A5/PI staining and then analyzed by flow cytometry (mean ± SD). (E) Example smoothed flow cytometry density plots. (F) Example single A5-FITC-positive necroptotic cells, imaged using live microscopy. (G–H) 3 x 10⁴ HaCaT cells per well in 96-well plate were stimulated, and PI and A5-FITC were added. The plate was placed on IncuCyteZOOM apparatus and 2 images per well were recorded every 30–45 minutes. (G) Normalized PI- or A5-positive objects per image. (H) Example single A5-positive necroptotic cells, imaged using IncuCyteZOOM apparatus. (I) HaCaT cells were harvested, and the cell death key factors pMLKL and CC3 were detected using western blot. Representative data are shown from 1 of at least 2 independent experiments. All raw data for the data summarized under this Fig can be found in S1 Data. A5, annexin V, CC3, cleaved caspase 3; Con, control; FITC, fluorescein isothiocyanate; MLKL, mixed lineage kinase domain-like; Nec-1s, necroptotic inhibitor of RIPK1; NSA, necrosulfonamide; PI, propidium iodide; pMLKL, phosphorylated mixed lineage kinase-like; RIPK1, receptor-interacting protein kinase-1; RIPK3, receptor-interacting protein kinase-3; Sd, protein ladder; SD, standard deviation; TS, TNFα + SMAC mimetic; TSZ, TNFα + SMAC mimetic + zVAD.