Measuring Cellular Bioenergetics under Hypoxia

Maximum glycolytic capacity and ATP production rate using methods reported by Mookerjee and colleagues [29 (link),30 (link)]. Briefly, 24 h prior to assay ShCtrl, ShBnip3 and cell with DFP were plated in 24-well Seahorse V7-PS assay plate under hypoxia. Prior to measurement cells were washed three times with 500 ul of Krebs Ringer Phosphate HEPES/KRPH (2 mM HEPES, 136 mM NaCl, 2 mM NaH₂PO₄, 3.7 mM KCl, 1 mM MgCl₂, 1.5 mM CaCl₂, 0.1% [w:v] fatty-acid-free BSA (Sigma, A7511), pH 7.4 at 37°C) and incubated 37°C for 1 h under 100% air. Oxygen consumption rate (OCR) and associated extracellular acidification rate (ECAR) were measured in a Seahorse XF-24 analyzer by addition via ports A-C of 10 mM glucose, 1 μM rotenone (Sigma, R8875) plus 1 μM myxothiazol (Sigma, T5580), and 200 μM monensin (Sigma, M5273) plus 1 μM FCCP (Sigma, C2920) for measuring glycolytic capacity. To measure ATP production rate from oxidative and glycolytic, OCR and ECAR was measured by addition of 10 mM glucose, 2 μg oligomycin (Sigma, 75,351), 1 μM rotenone plus 1 μM myxothiazol. The rate of oxygen consumption and extracellular acidification were normalized to protein content of the appropriate well.