Xf assay media

The oxygen consumption rate (OCR) in cells was measured using the XF24 extracellular flux analyzer (Seahorse Biosciences, North Billerica, MA), as previously described [14 (link)]. HCT116 cells in XF24 cell culture plates were incubated for 24 h (Seahorse Biosciences) and incubated for an additional 30 min at 37°C without CO₂ in XF assay media (pH 7.4, Seahorse Biosciences) containing various concentrations of compound 7. After determining the basal OCR, oligomycin (1 μM), carbonylcyanide p-trifluoromethoxyphenylhydrazone (0.5 μM), and rotenone (1 μM)/antimycin A (1 μM) were added sequentially, and the OCR was determined following each addition.

Cells were seeded overnight in 24‐well culture plate (SeaHorse Bioscience, Billerica, MA) at a density of 5 × 10⁵ cells/well, and incubated either under standard (n = 22 wells for control) or heat‐stressed conditions (n = 22 wells heat stress). Following the 24 h incubation, culture media was removed and replaced with XF Assay Media (SeaHorse Bioscience). Per manufacturers’ protocol, SeaHorse injection ports were loaded with oligomycin and carbonyl cyanide p‐[trifluoromethoxy]‐phenyl‐hydrazone (FCCP). Prior to any chemical treatment, basal oxidative metabolism (oxygen consumption rate, OCR) was measured. Then to reveal endogenous proton leak (mitochondrial uncoupling) cells were treated with oligomycin at a final concentration 1.0 μmol/L. Finally, FCCP, an uncoupler of electron transport at a concentration of 1.25 μmol/L was added to determine peak oxygen consumption (an indirect indicator of peak oxidative metabolism) (Giulivi et al. 2008; Wikstrom et al. 2012). SeaHorse XF24 Extracellular Analyzer was run using 8 min cyclic protocol commands (mix for 3 min, let stand 2 min, and measure for 3 min) in triplicate as previously performed (Vaughan et al. 2013).

Cells were plated at optimal densities in Seahorse XF 24-well plates one or two days prior to the measurement. Cells were incubated with Seahorse XF Assay Media at 37 °C for 1 h without CO₂ for basal OCR and with MAS buffer (Mannitol and sucrose buffer: 70 mM sucrose, 220 mM Mannitol, 10 mM KH2PO4, 5 mM MgCl2, 2 mM HEPES, and 1 mM EGTA in diH2O. pH 7.2 using 0.1 M KOH) to measure complex activity just before running the assay. Substrate concentrations were 1μM for Oligo and FCCP, 1μM/0.5μM for Rot/AA, and 5 mM for succinate, all the substrates were purchased from Seahorse Bioscience. Reagents for complex activity such as Saponin 100 μg/ml, Pyruvate 10 mM, Malate 2 mM, ADP 50 μM and NADH 10 mM were purchased from Sigma. OCR measurements were obtained using the Seahorse XFe24 Analyzer, and normalized to protein concentration (µg/µL).

Mouse primary SAT-derived SVF was isolated from WT and UCP1 KO mice and seeded into 0.2% gelatin (wt/vol) coated 24-well XF cell culture microplates (Seahorse Bioscience, Billerica, MA, USA). Differentiated SVF cells were treated with or without 100 μM EPA. Then, culture media were changed to the XF assay media (Seahorse Bioscience, Billerica, MA, USA) containing 2 mmol/L sodium pyruvate and 25 mmol/L glucose and placed in a 37 °C non-CO₂ incubator for 1 h. To determine oxygen consumption rate (ORC) changes in the differentiated SVF cells, an XF Cell Mito Stress Test Kit (Agilent, Santa Clara, CA, USA) was used and oligomycin A (1 mmol/L), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 0.3 mM), and antimycin/rotenone (A/R, 1 mM each) were injected according to the manufacturer’s instructions. Respiration profiles were calculated by the following formula: