Seahorse xf cell mito stress test

Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of CD4+ T cells were measured using a Seahorse XFe96 Analyzers. T cells were cultured with or without BMP4 (10ng/ml, PeproTech) for 24 hours, and then were prepared in XF RPMI medium supplemented with 10 mM glucose, 1 mM pyruvate and 2 mM L-glutamine, added 2 × 10⁵ T cells into each well of Seahorse XF96 cell culture microplates (Coated with 22.4 μg/ml Corning^® Cell-Tak Cell and Tissue Adhesive), spined down and preincubated at 37°C for around 30 min in the absence of CO², then ran the program of the Seahorse XF Cell Mito Stress Test.

Cells were plated at in XF96 microplates (Seahorse Bioscience, Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instruction and as previously described (29 (link)). For Seahorse XF Cell Mito Stress Test, 24 h post treatment medium was changed to XF base medium, supplemented with 1 mM pyruvate, 2 mM glutamine and 10 mM glucose, and incubated for 1 h at 37°C in a CO₂-free incubator. Mitochondrial oxidative phosphorylation on the basis of the oxygen consumption rate (OCR) and glycolysis by analyzing the extracellular acidification rate (ECAR) were estimated following oligomycin (LNCaP: 2 µM, HS5: 1 µM)), carbonyl-cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (LNCaP: 0.25 µM, HS5: 1 µM) and rotenone and antimycin A (both 0.5 µM) treatments at indicated time points using a Seahorse XFe 96 Analyzer. Hoechst 33342 (10 µg/ml, Thermo Fisher Scientific; Waltham, MA) was used for individual normalization to DNA.

Cells were seeded on Seahorse XFe24 culture plates (Agilent, Santa Clara, CA, USA); oxygen consumption (OCR) and extracellular acidification (ECAR) measurements were performed at 6 min intervals (2 min mixing, 2 min recovery, 2 min measuring) in a Seahorse XFe24 Extracellular Flux Analyzer (XFe Wave software). Consecutive treatments with oligomycin (1 µM final), FCCP (0.5 µM final), and rotenone/antimycin A (0.5 µM final) were performed to enable quantification of basal OCR, ATP-coupled OCR, proton leak, and maximal respiration (Seahorse XF Cell Mito Stress Test, Agilent). ATP production was quantified with ATP Detection Kit (Molecular Probes, Eugene, Oregon, USA) following manufacturer’s instruction.

WT and ΔMAVS J-Lat clones were seeded at 200,000 cells/well in CELL-TAK (Corning) coated XF96 Cell Culture Microplate. The Seahorse XF Cell Mito Stress Test was performed on XFe96 Analyzer according to manufacturer’s protocol (Agilent). OCR values for Basal, Proton Leak, ATP Production, Non-mitochondrial Respiration, Maximal Respiration, and Spare Respiratory Capacity were calculated with Seahorse XF Stress Test Report Generator within Wave software (Agilent).

Hepatocytes were plated in a collagen-coated (Sigma-Aldrich C3867) seahorse XF96 cell culture microplates at 5000 cells/well in 80 µL in Buffer C and incubated at 37 °C for 6 h. After 6 h media was changed, and hepatocytes were cultured overnight before any experimental procedure. Mitochondrial measurements and FAO were performed using the Seahorse XF Cell Mito Stress Test (Agilent 103015-100) according to manufacturer’s instructions. Briefly, primary hepatocytes were plated as indicated above and the following morning regular media was replaced for Seahorse XF DMEM medium (Agilent 103575-100) supplemented with 10 mM glucose (Agilent 103577-100), 1 mM Sodium pyruvate (Agilent 103578-100) and 2 mM glutamine (Agilent 103579-100). Oxygen consumption Rate (OCR) was measured following the sequential addition of 40 µM of Etomoxir (MedChemTronica HY-502002; port A), 1.5 µM of oligomycin (port B), 0.3 µM of carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP)(port C) and 0.5 µM of Antimycin/Rotenone (Rot/AA) (port C). The oligomycin, FCCP and the Rot/AA were purchase from Agilent (103015-100). Oxygen consumption rate (OCR) was normalized to protein concentration using BCA Protein Assay (Thermo Scientific™ 23222). All parameters were represented as normalize OCR.