Measurement of Mitochondrial O2 consumption

MitoXpress^® Xtra–Oxygen Consumption Assay (Luxel Biosciences) was used for the direct, real-time analysis of cellular respiration and mitochondrial function. MitoXpress^® Xtra is quenched by O₂ through molecular collision, and thus the amount of fluorescence signal is inversely proportional to the amount of extracellular O₂ in the sample. HUVEC were serum starved for 1 hour and incubated with 3% plasma for 4 hrs. After incubation cells were washed with clear respiration media. The MitoXpress-Xtra-HS probe was added to cells in accordance with manufacturer’s instructions. Oxygen consumption was measured using time-resolved fluorescence (TR-F) with a dual delay of 30 μs and 70 μs using a VarioSkan fluorescence plate reader (Thermo-Scientific). Rate of oxygen consumption was determined from the slope of fluorescence vs. time for each sample using relative fluorescence units/hour.