Cytochrome c Release Assay in MV4-11 Cells

MV4-11 cells were incubated at 5 × 10⁵/ml in culture medium for four hours with the indicated drug. Cytochrome c release (using Alexa-647-conjugated cytochrome c antibody, Becton Dickinson) was measured after a further 60 minute incubation of digitonin permeabilised cells with BH3 peptides as described [12 (link), 47 (link)]. Adjustments for peptide-induced cytochrome c release in untreated cells were made in order to establish agent-specific release (delta priming), using the formula 100X (percent cytochrome c positive with peptide – percent cytochrome c positive with drug plus peptide)/(percent cytochrome c positive with peptide). A mutated PUMA-BH3 peptide (PUMA2A) (Ryan et al. 2013) at 100 μM and BIM-BH3 peptide (10 μM) were used as controls in all experiments. Data were collected on a FACSCanto II flow cytometer (Becton Dickinson) and analysed with FACS Diva software (Becton Dickinson).