The compartment model correction avoids artifacts caused by O2 depletion in the chamber and in the walls. Basal respiration of cortical synaptosomes in Seahorse medium containing glucose plus pyruvate was recorded, followed by addition of oligomycin (2 μg/mL), FCCP (4 μM in the presence of BSA) and in (C and D) rotenone (2 μM) plus myxothiazol (2 μM), as indicated. (A and B) The experiment was designed according to the manufacturer’s recommended ideal conditions, not allowing [O2]M to deplete by more than 50 mmHg. Data points show the mean ± sem of values from 11 wells from four independent plates. (C and D) Double density plating of synaptosomes resulted in oxygen depletion during uncoupled respiration. Data points show the mean ± sem of values pooled from 55 wells from three independent plates. (B and D) Gray squares, noncorrected oxygen consumption rate calculated as mean −d[O2]M/dt for each measurement cycle; black diamonds, mean corrected OCR(t) calculated using the rate constants given in Table for polystyrene plates. Note that in ‘ideal conditions’ (B) noncorrected and corrected relative oxygen consumption rates overlap, but in (D) the noncorrected values substantially deviate from (A) or from the corrected OCR(t) (black diamonds). The noncorrected rates are overestimated because of (a) rejecting data points corresponding to very low [O2] and (b) O2 depletion in the walls.