Nonadherent cells were subjected to cytospin, fixed in 4% paraformaldehyde (PFA), and permeabilized with 0.1% Triton X‐100 for subsequent staining as described previously [37 (link)]. Single optical sections were acquired with an SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 63×/1.4 oil immersion objective lens. For the analysis of apoptotic Figures, cells were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and tetramethylrhodamine (TRITC) ‐conjugated Agglutinin (both from Thermo Fisher Scientific). For cytochrome c localization, cells were stained overnight at 4 °C with an antibody against cytochrome c (clone 7H8.2C12, Thermo Fisher Scientific) and counter‐stained with DAPI. Secondary staining was performed with an Alexa Fluor 488‐conjugated anti‐mouse antibody (Jackson ImmunoResearch, West Grove, PA, USA). The total and cytochrome c‐positive cellular areas were calculated from > 20 fields for each sample with an in‐house developed macro for imagej (RRID: SCR_003070). Briefly, the cytochrome c signal from each cell in the field of view was segmented using an automatic threshold (Otsu algorithm) and the area of the signal calculated on the binary images. The cytochrome c area was then normalized by the total cell area, which was identified on the bright‐field transmission image.
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