Fatty acid oxidation assay kit

The β-oxidation levels in PASMC were measure using the Fatty Acid Oxidation Assay Kit (Abcam, Cambridge, United Kingdom) and the OCR level were measured using the Seahorse system. Glutamine consumption level in cell medium were measured by using the Glutamine-Glo Assay Kit (Promega, Madison, WI) as an indicator of the glutaminolysis levels.

The FAO rate was determined using a Fatty Acid Oxidation Assay Kit (ab217602; Abcam) and Extracellular O₂ Consumption Assay Kit (ab197243; Abcam). Briefly, the cells were seeded in 96-well culture plates and cultured in glucose-free RPMI 1640 medium at 37 °C overnight. The culture medium was removed and replaced with 150 μL of assay medium containing oleate as the FAO substrate, followed by incubation at 37 °C for 30 min in a CO₂ incubator. The FAO rate was determined by measuring extracellular O₂ consumption (EOC) in assay medium. EOC was measured based on the ability of oxygen to quench the excited state of the kit reagent, which was added in a 10-μL amount (along with two drops of pre-warmed high-sensitivity mineral oil) to each well containing assay medium. The emitted fluorescence intensity at 650 nm after excitation at 380 nm was measured over 80 min using a plate reader (Molecular Devices, San Jose, CA, USA). The fluorescence values were summed. The FAO rate was determined by comparing MS-275- and DMSO-treated MPHs.