Wave desktop 2

Real-time OCRs of AMs, PMs and BMDMs were measured in XF medium (non-buffered RPMI containing 2 mM l-glutamine, 25 mM glucose and 1 mM sodium pyruvate) using a Seahorse Xfe 96 Analyzer (Agilent Technologies). For the mitochondrial stress test, mitochondrial inhibitors oligomycin (1.5 µM), fluorocarbonyl cyanide phenylhydrazone (FCCP) (1 µM), antimycin A and rotenone (0.5 µM) were used as per the manufacturer’s recommendations. In brief, cells were seeded at a density of 100,000 cells per well and 3 basal measurements were taken. Following this, two consecutive measurements were taken following each injection of oligomycin, FCCP and antimycin A with rotenone. All measurements were normalized to cell number using crystal violet dye extraction assay. Oxygen consumption curves, OCRs and ECARs were generated using Wave Desktop 2.3 (Agilent Technologies).

Real-time oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of macrophages were measured in XF media (non-buffered DMEM containing 2mM L-glutamine, 25mM glucose and 1mM sodium pyruvate) using a Seahorse XFe 96 Analyzer (Agilent Technologies). For the mitochondrial stress test, mitochondrial inhibitors oligomycin (1µM, Sigma), fluorocarbonyl cyanide phenylhydrazone (FCCP, 1.5µM, Sigma), antimycin A (0.5µM, Sigma) and rotenone (0.5µM, Sigma) were used. Briefly, macrophages were seeded at a density of 100,000 cells per well and 3 basal measurements were taken. Following this, 2 consecutive measurements were taken following each injection of oligomycin, FCCP, and antimycin A with rotenone. Basal respiration was determined as the last basal measurement minus the non-mitochondrial respiration rate, as determined by the last reading following antimycin A/rotenone injection. ATP production was determined as the decrease in OCR following oligomycin injection and coupling efficiency is determined by the ratio of ATP production to basal respiration. Finally, spare respiratory capacity was determined as the absolute increase in OCR after FCCP injection compared to basal respiration. All measurements were normalized to cell number using a crystal violet dye extraction assay. Oxygen consumption curves were generated using Wave Desktop 2.3 (Agilent Technologies).

OCRs were measured using a Seahorse XFp extracellular flux analyzer (Agilent Technologies) as per the manufacturer’s instructions. Briefly, cells (U1 or Primary CD4⁺ T cells) were seeded at a density of 10⁴–10⁵ per well in a Seahorse flux analyzer plate precoated with Cell-Tak (Corning). Cells were incubated for 1 hr in a non-CO₂ incubator at 37°C before loading the plate in the seahorse analyzer. To assess mitochondrial respiration, three OCR measurements were performed without any inhibitor in XF assay media to measure basal respiration, followed by sequential addition of oligomycin (1 μM), an ATP synthase inhibitor (complex V), and three OCR measurements to determine ATP-linked OCR and proton leakage. Next, cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; 0.25 μM), was injected to determine the maximal respiration rate and the SRC. Finally, rotenone (0.5 μM) and antimycin A (0.5 μM), inhibitors of NADH dehydrogenase (complex I) and cytochrome c - oxidoreductase (complex III), respectively, were injected to completely shut down the ETC to analyze nmOCR. Seahorse data were normalized to total amount of protein (μg) and mitochondrial respiration parameters were analyzed using Wave Desktop 2.6 software (Agilent Technologies).

Glycolysis stress test kit (Agilent technologies, 103020‐100) was used in order to measure extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Briefly, cells were plated at 4,000 cells per well cell density in XF96‐well plate pretreated with poly lysine. For silencing, cells were transfected using lipofectamine RNAimax as per manufacturer’s guidelines before plating. 48 h after transfection, cells were treated with 10 µM ENZA for 12 h. On the day of experiment, media were replaced with phenol‐red free Seahorse XF base medium (Agilent, 103335‐100) containing 2 mM L‐glutamine. 10 mM glucose, 2 µM oligomycin, and 50 mM 2‐DG were added in ports A, B, and C on indicated times. Data were normalized to cell number measured by crystal violet assay. For crystal violet assay, cells were sequentially incubated with 0.1% glutaraldehyde and crystal violet for 5‐15 min each. After washing excess stain, plate was dried. Finally, 100 µl of Sorenson’s solution was added to each well and absorbance taken at 570 nm. Data analysis was performed using Wave Desktop 2.6 software from Agilent technologies.