Figure 2. Optimized plasma membrane permeabilization by saponin does not compromise mitochondrial outer membrane integrity.

(A) Primary rat cortical neurons were control-treated (con, filled squares) or permeabilized by saponin (sap, 25 µg/ml) plus EGTA (5 mM) in aCSF medium in the absence (filled circles) or presence (open circles) of purified cytochrome c (100 µM) after three baseline O₂ consumption rate (OCR) measurements (first arrow). Pyruvate and malate (P/M, 5 mM each), ADP (1 mM), and excess K₂PHO₄ (3.6 mM for 4 mM final) were co-injected with saponin to measure complex I-dependent ADP-stimulated respiration. Subsequently, oligomycin (oligo, 0.3 µg/ml), FCCP, (F, 2 µM) and antimycin A (AA, 1 µM) were added as indicated (arrows). Pyruvate (10 mM) was included with FCCP for intact cells (filled squares). (B) Neurons were permeabilized as described in (A) using 250 µg/ml saponin in the absence (filled triangles) or presence (open triangles) of purified cytochrome c (100 µM). Control-treated neurons (filled squares) and neurons permeabilized by 25 µg/ml saponin (filled circles) are duplicated from (A) for comparison. OCRs in (A) and (B) are mean ± SD in quadruplicate, normalized to the third measurement point and expressed as % baseline OCR. All treatments in (A) and (B) were assayed in parallel on an individual 24-well plate.