Xf24 extracellular analyzer

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).

The oxygen consumption rate (OCR) was measured with an XF24 extracellular analyzer (Seahorse Bioscience, North Billerica, MA, USA). Briefly, 7.5 × 10³ HEK-1B1 cells were seeded in poly-l lysine-coated XF24 culture plates and cultured in a DMEM with 10% TH-depleted serum for 4 days. The cells were treated with T₃ (100 nM) or MGL-3196 (6 µM) 48 h before OCR measurement. Reagent concentration was optimized using a Mito Stress Test kit from Seahorse Bioscience (103015-100) according to the protocol and the XF24 program’s algorithm. Oligomycin, FCCP, antimycin A, and rotenone were all used at a concentration of 1 µM. On the day of measurement, the TH-depleted medium was replaced by 500 µL of the assay medium, and the plate was incubated at 37 °C for 1 h in a non-CO₂ incubator.
Basal respiration was calculated as [baseline O₂ consumption] − [OCR after rotenone and antimycin A]. ATP production corresponds to the OCR used for mitochondrial ATP synthesis via ATP synthetase, which is inhibited by oligomycin. The expression [OCR after FCCP] − [OCR after rotenone and antimycin A] determined the maximal respiration (respiratory capacity), and spare respiratory capacity was calculated as [OCR after FCCP] − [Basal OCR].