Xf plasmamembrane permeabilizer xf pmp

Recombinant, mutant perfringolysin O (rPFO; commercially XF Plasma Membrane Permeabilizer [XF PMP, Agilent Technologies]) was used to selectively permeabilize the plasma membrane of BMDMs. Experiments were conducted as previously described^{57 ,58 (link)}. Immediately prior to assay, cell media was replaced with MAS buffer (70 mM sucrose, 220 mM mannitol, 10 mM KH₂PO₄, 5 mM MgCl₂, 2 mM Hepes, and 1 mM EGTA; pH 7.2) containing 3 nM rPFO, respiratory substrates, and 4 mM ADP. The ADP-stimulated respiration rate (referred to as ‘State 3’ respiration) was measured, and rates were subsequently measured in response to 0.2 μM rotenone with 1 μM antimycin A. Substrate concentrations were as follows: Glutamate/Malate, 5 mM glutamate with 5 mM malate; Pyruvate/Malate, 5 mM pyruvate with 1 mM malate; Succinate/Rotenone, 5 mM succinate with 2 μM rotenone; Citrate, 5 mM citrate.
When permeabilized cells were treated with alamethicin to form pores of 3–6 kDa in the mitochondrial inner membrane (“double-permeabilized” cells) and complex I-mediated respiration was directly assessed, 10 μg/mL alamethicin was added at 37°C 15 minutes prior to measurements^{58 (link)}. Double-permeabilized cells were offered 10 μM cytochrome c in the experimental medium and either 10 mM NADH or 10 mM succinate with 2 μM rotenone to drive respiration.