Changes in mitochondrial function in DCs after TLR activation. (A) BM-derived DCs were seeded in a Seahorse XF-24 analyzer, stimulated with LPS, and at the indicated time points OCR was determined. Dashed line represents baseline OCR. Data represent means ± SD of triplicates. One representative experiment of 3 is shown. (B) DCs were seeded in a Seahorse XF-24 analyzer, stimulated with medium as a control or LPS for 24 hours, and real-time OCR was determined during sequential treatments with oligomycin (ATP-synthase inhibitor), FCCP, and antimycin-A/rotenone (ETC inhibitors). Data represent means ± SD of triplicates. One of 6 experiments is shown. (C-D) Twenty-four-hour LPS-stimulated DCs were stained with hydroethidin (HE) to detect O₂⁻ production or DiOC₆ to determine mitochondrial membrane potential (ΔΨ_m). A representative histogram is shown and the bar graph represents means ± SD of 3 independent experiments. **P < .01. (E) Twenty-one hours after stimulation with LPS or medium, DCs were fed ¹³C₆-glucose or normal ¹²C-glucose (both 10mM) for 3 hours. Next, metabolites were isolated from cells by methanol extraction and the ¹³C content in citrate was quantified by mass spectrometry. Amounts of citrate carrying 1-6 ¹³C are shown relative to levels of citrate harboring no ¹³C (only ¹²C). Relative amounts of naturally occurring ¹³C in citrate when DCs are pulsed with unlabeled glucose are shown in triangles. Data represent means ± SD of triplicates. ***P < .001 compared with naturally occurring ¹³C in citrate. Data represent means ± SD of triplicates of 1 experiment. (F) mtDNA/nDNA ratio at different time points after LPS stimulation. Data represent means ± SD of 2 independent experiments. ant/rot indicates antimycin-A/rotenone; and oligo, oligomycin.