Fig. 10.

Evaluation of complex I and II substrate linked activities in permeabilized cells. Seahorse extracellular flux analysis in PMP permeabilized primary cortical rat neurons exposed for 4 h to increasing concentrations of chloroquine (CQ). (A-B) ADP was added during permeabilization in the assay to determine OCR under ATP synthase limiting conditions. (C-D) FCCP was added during permeabilization in the assay to determine the maximum rate of respiration in the presence of substrates for each respective complex. Complex I substrate linked activities were measured by the addition of pyruvate (P) and malate (M) (calculated as OCR after these substrates minus OCR after rotenone (R). Complex II substrate linked activities were measured by the addition of succinate (suc) (calculated as OCR after substrates minus OCR after antimycin A (A)). Rotenone (R) was added to inhibit complex I before complex II measurements. Antimycin A (A) was added to inhibit respiration through electron transport chain. Shown is a representative experiment with data = mean ± SEM n ≥ 3 independent samples, p < 0.05.