Mitochondrial Respiration Profiling in HEK001 Cells

Mitochondrial oxygen consumption rate (OCR) was measured using a XFe24 analyzer (Seahorse Bioscience, North Billerica, MA, USA) as previously described [29 (link)]. Briefly, HEK001 cells were cultured on a XF24 V7 plate to give 7 × 10³ cells/well after the HEK001 cells had been kept in normal growth medium for 24 h. The medium was then replaced by hesperetin containing growth medium for 48 h. After hesperetin treatment for 48 h, the culture medium was replaced by fresh normal growth medium for 1 h before OCR measurement. The OCR was measured at 37 °C before and after adding the indicated chemicals (1 μM oligomycin A [an ATP synthase inhibitor] and 0.5 μM rotenone [a mitochondrial complex I inhibitor] with 0.5 μM antimycin A [a mitochondrial complex III inhibitor]). This was in order to monitor the OCR contributed by mitochondrial basal respiration and ATP-linked respiration, and by non-mitochondrial respiration [30 (link)]. The results are presented in pmol/minute/μg protein.