Apoptosis assay

Totally, 5 × 10⁵ cells/ml DL-BCL cells were treated for 24 h with increasing concentrations of either BDA-366/venetoclax or a single concentration of venetoclax/BDA-366 or the combination of both treatments. Afterwards, cells were pelleted by centrifugation, and incubated with Annexin V-FITC (Life Technologies, Brussels, Belgium) and 7-aminoactinomycin D (7-AAD) (Life Technologies, Brussels, Belgium) or with 2.5 µM NucviewTM 488 caspase-3 substrate (Biotium, CA, USA) for 15 or 30 min, respectively. Cell suspensions were analyzed with an Attune^® Acoustic Focusing Flow Cytometer (Applied Biosystems, Brussels, Belgium). Cell death was scored by quantifying the population of Annexin V-FITC-positive and 7-AAD positive cells or by quantifying the caspase-3 positive cells. After treating the BMK cells (3000 cells/well in 384-well plate) for 24 h with BDA-366, cells were stained for 30 min with DRAQ5, TMRE and Annexin V-Alexa fluor 488. Images were taken using the Opera High Content Screen System (PerkinElmer) with ×20 air objective. Intensity and morphology features were extracted from the fluorescent images through image segmentation and analyzed using Acapella analysis software (PerkinElmer) script (available for free at http://www.andrewslab.ca). Quantitative analyses of cell death results in cell lines are from cell populations in which at least 80% of the cells were viable in control conditions.