IFNγ Increases Monocyte OCR. (A-B) Real-time changes in ECAR (A) and OCR (B) measured using Seahorse extracellular flux analyzer in primary human monocytes immediately following injection of media alone, LPS, IFNγ, or LPS plus IFNγ (n = 5 technical replicates). (C-D) Primary human monocytes were stimulated with media alone, LPS, IFNγ, or LPS plus IFNγ for 24 hours prior to the start of the assay, then (C) ECAR was measured according to the Seahorse Glycolysis Stress Test or (D) OCR was measured according to the Seahorse Mito Stress Test. For the Glycolysis Stress Test, cells were sequentially treated with glucose (10 mM), oligomycin (1 µM), and 2-deoxyglucose (50 mM). For our modified assay, cells were treated with PMA (100 µM), then, in some cases, rotenone and antimycin A (0.5 µM). For the Mito Stress Test, cells were sequentially treated with oligomycin (2 µM), FCCP (0.5 µM), and rotenone+antimycin A (0.5 µM). OCR and ECAR were measured in a standard, 6-minute cycle. Data displayed in panels A-D are representative tracings of 3 independent experiments. (E-F) Modified Seahorse Assay: primary human monocytes treated with IFNγ or media alone for 24 hours prior to the start of the assay (n = 5 technical replicates). Basal ECAR (E) and OCR (F) measurements were collected, then PMA was injected to stimulate maximal activation, followed by rotenone/antimycin A injection to inhibit mitochondrial oxygen consumption. Real-time changes in ECAR (E) and OCR (F) were monitored. (G) Significant differences in OCR measurements from panel F are displayed, in which the last measurement before PMA injection is used to quantify “basal” OCR, and the last measurement before rotenone/antimycin A injection is used to quantify “PMA-stimulated” OCR throughout the study. Data in panel G were analyzed by 2-way ANOVA with Sidak’s multiple comparisons test. Error bars are mean ± standard error of the mean. **P < .01, ****P < .0001.