4.5. Analysis of Cellular Bioenergetics Using Seahorse Extracellular Flux Technology

To measure mitochondrial function and glycolysis in isolated washed human platelets, a Seahorse XFe96 Analyzer was used as described previously [11]. Briefly, freshly isolated platelets were suspended in assay medium (bicarbonate-free low buffered DMEM supplemented with glucose (1 g/L) and glutamine (2 mM), pH 7.4), and introduced into the Seahorse XFe96-well plate (10 × 10⁶ platelets in 80 µL per well), followed by centrifugation (700 g, 5 min) and incubation in air without CO₂ (1h, 37 °C). Just before the start of measurement, either a control assay medium, an assay medium saturated with CO or an assay medium containing CORM-A1 were added to wells in volumes of 120 µL. Followed by 12 min of equilibration, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were assessed over time. In experiments on platelets treated with BAYs, the compounds were added from the ports after three initial measurements. The data presented in the figures were taken 30 min after addition of tested compounds.