LPS-activated DCs commit to glycolysis in response to NO-induced inhibition of mitochondrial respiration, but do not require sustained glycolysis for normal activation. (A) DCs were seeded in a Seahorse XF-24 Analyzer and either left unstimulated or treated with LPS for 24 hours in the presence or absence of SEITU, after which real-time rates of ECAR as a readout for lactate production were determined. Data represent means ± SD of 4 independent experiments. (B-C) DCs were treated as in panel A and supernatants collected 24 hours later were used to determine glucose consumption (B) and lactate production (C). Data represent means ± SD of 4 independent experiments. (D) DCs were seeded in a Seahorse XF-24 Analyzer and either left unstimulated or treated with the indicated reagents for 10 minutes, after which real-time rates of ECAR as a readout for lactate production were determined. Data represent means ± SD of 3 independent experiments. (E-F) DCs were stimulated with LPS for 24 hours in the presence or absence of the NOS inhibitor SEITU after which surface expression of indicated markers was analyzed by FACS (E) or cytokine levels (F) were determined in supernatants. Data represent means ± SD of 4 experiments. (G) Wild-type or iNOS^−/− DCs were treated for 6 hours with the indicated reagents and subsequently cultured for 4 days in a 1:10 ratio with CFSE-labeled naive OT-II or OT-I T cells. One of 3 experiments is shown. *P < .05; **P < .01; ***P < .001. ant/rot indicates antimycin-A/rotenone; and oligo, oligomycin.