Seahorse xfe96 technology

Mitochondrial oxygen consumption rate (OCR) was measured using a Mito Stress Test (31 (link)) and Seahorse XFe96 technology (Agilent Technologies, Santa Clara, CA). Naïve and IL-12-differentiated CD4⁺ T cells were seeded on Cell-Tak (Corning, Corning, NY)-coated 96-well Seahorse plates in triplicate at a density of 3 x 10⁵ cells/well within 200 ul, at a concentration of 1.5 million cells/ml, and maintained under standard cell culture conditions (5% CO₂, 37⁰C). Cells were incubated in XF Assay Medium (pH 7.4) with sodium pyruvate (1 mM), L-glutamine (2 mM), and glucose (10 mM) at 37°C without CO₂ for 1 hr before measuring T cell OCR with an XFe96 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA) according to manufacturer’s instructions. Respiratory parameters were assessed by the sequential addition of oligomycin (1 μM), carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP; 2 μM), and rotenone/antimycin A (0.5 μM). OCR measurements were normalized to cell count obtained by staining nuclei with Hoescht dye (2 μM; ThermoFisher Scientific) and visualizing on a BioTek Cytation 1 cell imaging multi-mode reader (BioTek, Winooski, VT).

Mitochondrial oxygen consumption rate (OCR) is measured using a Mito Stress Test and Seahorse XFe96 technology (Agilent Technologies, Santa Clara, CA) as previously published [33 (link)]. Briefly, myoblasts are seeded in triplicate on a collagen-coated 96-well Seahorse plate (35,000 cells/well) and maintained under standard cell culture conditions. After 24 h, growth media is replaced with XF Assay Medium (pH 7.4) with sodium pyruvate (1 mM), l-glutamine (2 mM), and glucose (10 mM) and incubated at 37 °C without CO₂ for 1 h before measuring myoblast OCR. Respiratory parameters are assessed by the sequential addition of oligomycin (1.5 µM), carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP; 2 μM), and rotenone/antimycin A (0.5 μM). Resulting OCR measurements are normalized to cell count obtained by staining nuclei with Hoechst dye (2 µM; ThermoFisher Scientific, Waltham, MA) and visualizing on a BioTek Cytation 1 cell imaging multi-mode reader (BioTek, Winooski, VT).