Intracellular BH3 Profiling of Lung Fibroblasts

Intracellular BH3 profiling was performed as described (77 (link)) on primary lung fibroblasts. Single-cell suspensions were obtained from enzymatically dispersed naive and fibrotic lungs. Lin^– cells were column enriched by incubating with CD45, CD31, and CD326 MicroBeads and purified off LS columns per manufacturer’s instructions (Miltenyi Biotec). Lin^– cells were stained with Live/Dead (1:100, L34965, Invitrogen) and anti-PDGFRα, -CD31, -CD45, and -EPCAM (1:100, Invitrogen, Thermo Fisher Scientific) for 30 minutes. Cells were permeabilized with 0.002% digitonin in MEB2 buffer (150 mM mannitol, 10 mM HEPES-KOH, 150 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1% BSA, 5 mM succinate in sterile distilled H₂O at a pH of 7.5) and exposed to BIM or BMF (100 μM, New England Peptides), DMSO, or 25 μM alamethicin (Enzo) for 60 minutes. Cells were fixed with 4% paraformaldehyde for 10 minutes, then solution neutralized (1.7 M Tris base, 1.25 M glycine, pH 9.1), stained with anti–cytochrome c (1:40, 11-6601-82, Invitrogen) in intracellular staining buffer (2% Tween 20 and 0.1 g/mL BSA in PBS) overnight at 4°C followed by FACS analysis on the LSRFortessa, and analyzed with FlowJo 2 software. Depolarization (% cytochrome c loss) was determined by measuring median fluorescence intensity (MFI) of the FITC (cytochrome c) gate and adjusting for positive and negative controls: depolarization = 1 – (MFI_sample – MFI_alamethicin)/(MFI_DMSO – MFI_alamethicin) (77 (link)).