Extracellular Acidification Rate in RBC

The ECAR was measured in a Seahorse XFe24 analyzer as previously reported with slight modifications [15 (link)]. For RBC samples, 5 × 10⁶ cells per well were plated onto XF24 cell plates coated with Cell-Tak (354242, Corning, New York, USA) in XF RPMI assay medium pH 7.4 (100 μL, XF assay medium) supplemented with glucose (40 mM), sodium pyruvate (1 mM) and L-glutamine (2 mM) and left for 10 min at room temperature. After seeding, the cell plates were centrifuged for 1.5 min at 200 × g at room temperature with an acceleration of one without braking. Afterward, 400 μL of XF assay medium was added to the cells, and the cells were incubated for 30 min at 37 °C without CO₂. XF measurements included serial injections of glucose (10 mM), oligomycin A (5 μM) and 2-deoxy-glucose (2-DG, 50 mM).