For the analysis, we defined a segmentation based on nuclei staining with Hoechst 33342. Then we established four groups in order to classify five populations: (1) Live cells were defined as negative cells for PI and normal staining with Hoechst 33342; (2) Apoptotic cells were defined as negative cells for PI and nuclei with increased staining with Hoechst 33342 (due to DNA condensation); (3) Necrotic cells were defined as positive for PI and negative for FLICA 660-YVAD-FMK staining; (4) pyroptotic cells were defined as double positive for PI and FLICA 660-YVAD-FMK. Twenty fields were acquired for each well. Four different experiments were performed and approximately 2000–10,000 cells were analyzed per experimental condition. The analysis was performed in the In Cell Analyzer 1000 Workstation software using the Multi Target Analysis Module.
