2.7. Measurement of whole-cell oxygen consumption rate

Using the Seahorse XF96 (Seahorse Bioscience), oxygen consumption rate (OCR) was measured essentially as described previously [12]. Briefly, H4IIE cells were plated at a density of 3 × 10 [4] cells/well in 80 μl serum-containing medium and incubated overnight. For the assay, cells were washed twice and incubated for 2 h in 200 μl serum-free medium (Phenol red free DMEM [A14430 Gibco], 25 mM HEPES, 5.5 mM glucose, 2 mM L-Glut, and 2.5 mM pyruvate). 180 μl fresh media was added and the plate was degassed at 37 °C for a further 1 h. OCR was continuously measured for a period of 50 min. 20 μl of 10X stock drugs made up in the same SF media were added after the first baseline reading.