Alexa 647 conjugated cytochrome c antibody

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).

Cells were incubated at 5x10⁵/ml in culture medium for four hours with the indicated drugs. Cytochrome c release (using Alexa-647-conjugated cytochrome c antibody, Becton Dickinson #558709) was measured after a further 60 minute incubation of digitonin-permeabilised cells with PUMA-BH3 peptide as described.[22 (link), 24 (link)] In preliminary assays, the PUMA-BH3 was optimised to 3 μM in all cells except M-091, as this was the concentration of the peptide found to be sufficient to induce mitochondrial outer membrane permeabilisation in drug-primed cells, but not so high that it induced a high degree of mitochondrial outer membrane permeabilisation without drugs. In M-091, the peptide was used at 0.5 μM. Adjustments for peptide-induced cytochrome c release in untreated cells were made in order to establish agent-specific release, 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) [22 (link)] 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).