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Measurement of oxygen consumption rate (OCR)

CH Chunrong Huang
LX Lian-Fang Xue
BH Bo Hu
HL Huan-Huan Liu
SH Si-Bo Huang
SK Suliman Khan
YM Yu Meng
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The OCR of cells was quantified employing a Seahorse XFe24 Analyzer (Agilent, MA, USA). Briefly, after treatment, the cells were washed, incubated with XF DMEM and D-glucose, and incubated in a non-CO2 incubator for 30 min at 37 °C. The sequential additions of oligomycin with a concentration of 1 μM (ATP synthase inhibitor), FCCP with a concentration of 2 μM (mitochondrial oxidative uncoupler), and rotenone with a concentration of 1 μM (mitochondrial electron transport chain complex I inhibitor) was done to assess the ATP production, maximal respiration, and spare respiratory capacity employing Seahorse Wave software (http://www.seahorsebio.com). The membrane potential was also determined using Cellular Membrane Potential Assay Kit (Fluorometric-Red) (ab176765) based on manufacturer's instructions provided.

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