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Seahorse assay

YX Youqin Xu
WC Wancheng Chen
JL Jing Liang
XZ Xiaoqi Zeng
KJ Kaiyuan Ji
JZ Jianlong Zhou
SL Shijun Liao
JW Jiexian Wu
KX Kongyang Xing
ZH Zilong He
YY Yang Yang
QL Qianzhen Liu
PZ Pingyi Zhu
YL Yuchang Liu
LL Li Li
ML Minfeng Liu
WC Wenxiao Chen
WH Wenhua Huang
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To measure the levels of glycolytic ATP production, 1 × 104 cells were seeded into each well of black 96-well plates. To normalize the levels of protein, the same number of cells were seeded into clear bottom 96-well plates. Cells were incubated in medium containing 1 μM oligomycin (Sigma-Aldrich) to inhibit mitochondrial oxidative ATP production or 25 mM 2-deoxy-D-glucose (2-DG) to inhibit glycolytic ATP production. After washing the cells with PBS, ATP levels were measured using a kit according to the manufacturer’s protocol (PerkinElmer). Total ATP production was calculated by subtracting the amount of ATP in cells treated with both oligomycin and 2-DG from the amount of ATP in cells without treatment. To normalize the number of cells, the protein concentration was measured using the Bicinchoninic Acid Protein Assay Kit (BCA, Sigma-Aldrich) according to the manufacturer’s protocols.

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