Cell Death Quantification Protocols

Short-term cell death was determined with an Incucyte FLR or Zoom imaging system (Essen Bioscience)⁵⁸ . Cells were treated as indicated in the Figure legend together with 30 nM Sytox green and imaged every 1 or 2 h. Analysis was performed using the Incucyte software and the number of dead (Sytox green positive) cells was normalised to the confluency at t = 0. Alternatively, cells were pre-treated for 1 h with 50 nM Syto 21, followed by cytotoxic treatments as indicated together with 5 μg/ml propidium iodide. Long-term colony formation assay was performed by plating 1000 cells per 6 well and treatment as described in the Figure legend. After 48 h of treatment with supernatant, the medium was changed to 2.5 μM venetoclax/S63845. Medium was changed to regular growth medium after an additional 48 h, and resulting colonies were stained with crystal violet after an additional 5 days. Cell death analysis via FACS was performed using Annexin V - propidium iodide staining^{59 (link)}. In short, treated cells were harvested and stained with 5 μg/ml propidium iodide and Annexin V (Biolegend) in Annexin V-binding buffer (10 mM Hepes pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) for 15 min. Flow cytometry was conducted on a BD FACSCalibur machine with BD CellQuest software and analysed using Flowing software; cells negative for propidium iodide and Annexin V were considered alive.