Seahorse assay

To measure cellular bioenergetics using extracellular flux, a Seahorse XF24 Extracellular Flux Analyzer (Agilent) was used. Cells were plated in XF 24-well cell culture microplates at a cell density of 5 × 10⁴ cells per well (Seahorse Bioscience) and incubated overnight. Next day, cells was replaced with unbuffered RPMI-1640 assay medium supplemented for 1-h equilibration. The OCR was measured over time at 6-min intervals. The first three measurements were conducted to establish a baseline rate, followed by three measurements after the addition of 2 μM oligomycin, to determine ATP turnover and the degree of proton leakage. Next, the maximal respiratory capacity was measured after the addition of the electron transport chain decoupler (FCCP, 0.5 μM). Finally, we administered 5 μM antimycin/rotenone to inhibit the flux of electrons through complex III and prevent oxygen consumption by the cytochrome c oxidase in the mitochondria. The value of OCR was determined by plotting the oxygen tension of the medium in the chamber as a function of time and normalized to total cell number. Absolute values of OCR were expressed as pmol per minute. The data were analyzed by the Seahorse Wave Desktop Software (version 2.4).