Measurement of mitochondrial oxygen consumption rate

the oxygen consumption rate (OCR) was measured by the Extracellular flux analyzer XF24 (Seahorse Bioscience, Houston, TX, USA). BV-2 cells were plated at 4 × 10⁵ cells/well in a Seahorse 24-well V7 microplate (Seahorse Bioscience) and cultured in normal high glucose DMEM growth medium (with NaHCO₃, glutamine, penicillin/streptomycin and 10% FBS, pH 7.4) for 24 h in a 5% CO₂ incubator at 37°C. Then, the medium was removed and cells were incubated in XF assay medium in the absence of NaHCO₃ and FBS for 1 h at 37°C in measuring chamber without CO₂ input according to the routine assay procedure of manufacturer's protocol. ATP, nigericin, ionomycin and the mitochondrial complex inhibitors [oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), rotenone and antimycin A] were freshly prepared in XF24 assay media. When determining the effect of acidification, high KCl and A438079 on OCR, medium of pH 4.5, pH 6.0, containing KCl (130 mM) or A438079 (10 μM) was added into wells prior to inserting the plate into the Seahorse XF24 Extracellular Flux analyzer. After 26 min to measure the basal respiration, nATP, aATP or nigericin was injected from port A; oligomycin (2.5 μM) was injected into each well at 50 min from port B, by FCCP (1 μM) at 74 min from port C and rotenone (2.5 μM) plus antimycin A (2.5 μM) at 98 min from port D. Because oligomycin is an inhibitor of ATP synthase by blocking the flow of protons through the F₀ subunit, the decreased OCR after oligomycin treatment indicates the status of ATP turnover. FCCP is an uncoupling agent which can disrupt ATP synthesis by transporting hydrogen ions across the mitochondrial membrane instead of the proton channel of ATP synthase (Complex V). This leads to a rapid consumption of energy and oxygen without the generation of ATP. Rotenone and antimycin A co-treatment can completely interfere with the electron transport chain in mitochondria. Rotenone inhibits the transfer of electrons in complex I to ubiquinone, while antimycin A blocks the electrons from complex III. OCR was recorded as pMoles per minute, and in some cases it was calculated as percentage of the OCR value before the treatment of tested agents as we previously described [64]. All the measurements and calculations were obtained by using seahorse instrument software (seahorse bioscience) according to the manufacturer's protocol. Averages of three wells were taken per data point.