2.12. Measurement of mitochondrial oxygen consumption rate

The oxygen consumption rate (OCR) was measured by the extracellular flux analyzer Seahorse XF24 (Agilent technologies, CA, USA) as we previously described [36]. Cells were plated at 4 × 10⁵ cells/well in a Seahorse 24-well V7 microplate and cultured in complete DMEM growth medium for 24 h in a 5% CO₂ incubator at 37 °C. Then, the medium was removed and the cells were incubated in XF assay medium in the absence of FBS for 1 h at 37 °C in measuring chamber without CO₂ input. The mitochondrial complex inhibitors such as oligomycin, FCCP, antimycin A1, and rotenone were freshly prepared in XF assay media, and their treated concentrations were 2.5 μM, 1 μM, 2.5 μM, and 2.5 μM, respectively. OCR was recorded as pMoles per minute. ATP turnover was measured after the treatment with oligomycin. Respiratory capacity was the maximum rate after the treatment of FCCP injection subtracted by the non-mitochondrial respiration.