PMA-stimulated OCR measures NADPH oxidase activity. (A-B) OCR was measured by Seahorse in primary human monocytes treated with media alone or IFNγ for 24 hours prior to the start of the assay (n = 5 technical replicates). (A) Monocytes from 4 healthy controls (black and green lines) and 4 CGD patients (red lines) were compared. (B) Monocytes were treated with (red lines) or without (black and green lines) DPI (100 uM) in addition to IFNγ or media alone for 24 hours. All OCR values were normalized by staining cells with 2 μg/mL Hoechst 33342 (ThermoFisher Scientific) for 30 minutes. Cells were then imaged and counted using the Biotek Cytation1. Cell counts were calculated by cell imaging software (Agilent) and imported into Wave (Agilent) using the normalization function. Raw OCR values were normalized by cell counts per well. (C) Bar graphs displaying PMA-stimulated OCR values normalized to the basal OCR levels in healthy control plus medium samples run in the same experiment (data not shown). Data are representative of at least 3 independent of experiments and were analyzed by 1-way ANOVA with Tukey’s multiple comparisons test. Error bars are mean ± standard error of the mean. ****P < .0001.