Standard plate reader

Oxygen consumption rate (OCR) were measured using the Seahorse XF24 and XF96 instruments (Seahorse Bioscience) under standard conditions and after addition of 0.1 μM oligomycin, 0.05 μM FCCP, 1 μM of rotenone and 10 μM of atpenin A5. Real time measurements (triplicates) of OCR in pMoles per minutes in tissue culture medium above cells plated in a microplate were plotted over time prior addition of rotenone to the culture media (basal oxygen consumption rate), and after addition of rotenone to specifically measure mitochondrial respiration. The difference in OCR prior and after rotenone addition to the culture media reflects the oxygen consumption by mitochondria (mitochondrial OCR). The OCR measurements were adjusted to cell numbers plated. To this end, the cells were stained with crystal violet (0.1% weight/volume of water) following paraformaldehyde (PFA) permeabilization (4% PFA) and spectrophotometric measurements of 10% acetic acid-solubilized cells were performed with a standard plate reader (Molecular Device). For OCR measurements of permeabilized cells, the Seahorse plasma membrane pemeabilizer kit was used, allowing cells to permeabilize for 30 min, with 10 mM malate and 10 mM glutamate supplemented media, and with sequential injection of 4 mM ADP, 2 μM rotenone, 10 mM succinate and 2.5 μM antimycin A.