Measuring Cellular Respiration Dynamics

Oxygen consumption rate (OCR) was determined using the Seahorse XF96 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA, USA) as previously described^{63 (link)}. Briefly, 2 × 10⁴ MCF10A-ras cells per well were seeded overnight in XF96 well culture microplates in a 37 °C /5% CO2 incubator. One hour prior to assay, cells were equilibrated with unbuffered DMEM and incubated at 37 °C for pH and temperature stabilization in a non-CO2 incubator. Analyses were performed both at basal conditions and after injection of Oligomycin (1 μM), FCCP (0.6 μM), Antimycin A/rotenone (1 μM each) at indicated time points. All data were analyzed using XF software and displayed as average OCR (pM/min).