Analysis of OCR and ECAR.

Cells were seeded at a density of 10,000 cells/well in RPMI medium in a 96-well plate. A total of five replicate wells per cell strain were plated. The next day, cells were washed 3 times with test medium, consisting of unbuffered, phenol red-free RPMI medium (Sigma), 2% FBS, 1 mM glutamine, and antibiotics. The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were measured using a Seahorse XFe96 extracellular flux analyzer.

To normalize OCR and ECAR to cell number, standard curves of cells (0 to 20,000) of each strain were plated in parallel tissue culture plates and stained with crystal violet. OCR and ECAR values were calculated using Seahorse Wave software.