Bboxiprobe r01 kit

L-15 leibovitz cell culture medium and Dulbecco’s modified eagle medium (DMEM) with high glucose were from HyClone (Logan, Utah, USA); FBS was obtained from Invitrogen (Carlsbad, CA, USA); antibiotics (penicillin, streptomycin, and amphotericin B), PBS, Percoll and trypan blue were from Solarbio (Beijing, China); sodium acetate, sodium propionate, sodium butyrate, and trichostatin A were purchased from Sigma (St. Louis, MO, USA); antibody against HIF-1α, dimethyl-bisphenol A and chrysin were from Santa Cruz (Santa Cruz, CA, USA); BBoxiProbe™ R01 kit was from BestBio (Shanghai, China); RNAeasy™ animal RNA isolation kit, reactive oxygen species assay kit and DAF-FM DA were purchased from Beyotime (Shanghai, China); HiScript^® III RT SuperMix for qPCR was from Vazyme (Nanjing, China); SYBR green qPCR kit was from Accurate Biology (Hunan, China); Pertussis toxin was obtained from APE×BIO (Houston, Texas, USA); Lysozyme assay kit was from Jiancheng (Nanjing, China).

Real-time O₂ consumption was measured using the BBoxiProbe™ R01 kit (Bestbio, China; BB-48211) according to the manufacturer’s instructions. The O₂ fluorescent probe was first diluted with PBS buffer to form an O₂ fluorescent probe staining working solution. C3H10T1/2 cells were treated with or without OLZ, the medium was removed, washed 3 times with PBS, 150 µl of fresh medium was added, 10 µl of O₂ fluorescent probe staining solution was added to each well and mixed thoroughly, 100 µl of oxygen blocking solution was added to each well, and fluorescence intensity was measured with an Operetta CLS (Perklin Elmer, Germany). Separate negative and positive controls were set up, i.e., untreated cells with antimycin (1 μM) and carbonyl cyanide p-trifluoromethoxy phenylhydrazone (FCCP, 5 μM). The oxygen consumption rate was calculated based on the change in fluorescence signal over 60min as follows: oxygen consumption rate (%) = (final fluorescence of OLZ-treated cells - initial fluorescence of OLZ-treated cells)/(final fluorescence of control cells - initial fluorescence of control cells) × 100%.