Fluorocarbonyl cyanide phenylhydrazone fccp

The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using an Extracellular Flux Analyzer XFp (Agilent Technologies, Santa Clara, CA, USA). Seahorse XF RPMI Base Medium (Agilent Technologies, Santa Clara, CA, USA; Cat#103336-100) replaced culture media; 200,000 T cells were seeded onto a Poly-D-Lysine (Gibco, Waltham, MA, USA; #A3890401) coated Seahorse 8-well plate and pre-incubated at 37 °C for 60 min in the absence of CO₂. To measure mitochondrial respiration and glycolysis, we used the XFp Mito Stress Test kit (Agilent Technologies, Santa Clara, CA, USA; #103010-100). Cells were resuspended in XF assay media supplemented with 25 mM glucose, 2 mM L-glutamine, and 1 mM sodium pyruvate. The OCR rate (pmoles/min) and ECAR (mpH/min) were measured at baseline and in response to 1 μM oligomycin, 1.5 μM fluorocarbonyl cyanide phenylhydrazone (FCCP), and 0.5 μM rotenone/antimycin A. All chemicals were purchased from Seahorse Bioscience (Agilent Technologies, Santa Clara, CA, USA). Cell counts between wild type and knockout T cells acquired by an Olympus automated cell counter were used to normalize the data. Calculations for individual parameters represent the average of individuals for each assay group. Error bars were calculated based on the individual well calculation for each parameter.

For the FAO-associated oxygen consumption rate (OCR) assay by Seahorse XFp Analyzer (Agilent), Palmitate-BSA reagent (Agilent) and Mito Stress Test Kits (Agilent) were used according to the manufacturers’ instructions with minor modifications. In brief, cells were collected and adhered to poly-D-lysine-coated XFp plates (4 to 6 × 10⁵ cells/well) via centrifugation in serum-free XF Base Medium (Agilent). For evaluation of palmitate oxidation, cells were cultured with XF Base Medium (Agilent) in a non-CO₂ incubator for 15–20 min at 37°C. Subsequently, the XF Palmitate-BSA FAO Substrates (Agilent) palmitate-BSA (167 μM palmitate conjugated with 28 μM BSA) and 0.5 mM L-carnitine (Sigma Aldrich) were added to the medium for the assessment of OCR. Immediately, XF Cell Mito Stress Test was performed in a Seahorse XFp Analyzer (Agilent) by sequentially adding several modulators of mitochondrial function, including 1 mM oligomycin (Oligo) (Agilent), 1.5 mM fluorocarbonyl cyanide phenylhydrazone (FCCP) (Agilent), and 100 nM rotenone plus 1 mM antimycin A (Rot/Ant A) (Agilent). Data were analyzed with Wave software version 2.3.0 (Agilent) and proper statistical methods using GraphPad Prism version 6 (GraphPad Software).

2 × 10⁵ BMDMs were plated into each well of Seahorse XF96 cell culture microplates (Agilent Technologies) and cultured overnight before treated with or without 20 ng/ml IL-4 for 6 or 24 h or ACs for 3 h. Basal and IL-4- or AC-induced changes in oxygen consumption rate (OCR) were measured with a Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies) using a Seahorse mito stress test kit. Extracellular acidification rate (ECAR) in BMDMs was recorded using a glycolysis stress test kit (Agilent Technologies). OCR and ECAR were measured under basal conditions and following the sequential addition of 10 mM glucose, 1 μM oligomycin, 1.5 μM fluoro-carbonyl cyanide phenylhydrazone (FCCP), 100 nM rotenone plus 1 μM antimycin A, or 50 mM 2DG (all the compounds were from Agilent Technologies), as described in the figure legends. After the assay, 3 μM Hoechst (Life Technologies) was added to each well to stain nuclei for cell counting. Results were collected with Wave software version 2.6 (Agilent Technologies). Data were normalized to cell numbers.