Ique flow cytometer

BH3 profiling was performed as described previously^{61 (link)}. Cells were suspended in MEB2 buffer (150 mM mannitol, 10 mM HEPES-KOH (pH 7.5), 150 mM KCI, 1 mM EGTA, 1 mM EDTA, 0.1% BSA and 5 mM succinate), added to a 384-well plate and incubated at 25 °C for 60 min combined with different BH3-only peptides in 0.001% digitonin for permeabilization. Cell were fixed with 4% formaldehyde for 10 min at room temperature followed by neutralization by adding N2 buffer (1.7 M Tris base, 1.25 M glycine, pH 9.1). Cells were stained overnight at 4 °C with Hoechst 33342 (H3570, Invitrogen) and anti-cytochrome c-Alexa Fluor 488 (6H2.B4; 612308) and analyzed using an Intellicyt iQue flow cytometer to determine the rate of loss of cytochrome c in response to each BH3 peptide. Assays were conducted in triplicates and P values were calculated using the Holm–Sidak method for multiple comparison t-tests, with α = 0.05. Dimethylsulfoxide and alamethicin were used as negative and positive controls for cytochrome c release. All assays were performed in triplicate.