Free Hb levels were measured in plasma/supernatant through spectrophotometry,23 (link) followed by the Allen correction. In order to examine the osmotically induced hemolysis, RBC were exposed to solutions of increasing saline (NaCl) concentration and the mean corpuscular fragility (MCF, concentration of NaCl at 50% hemolysis) was calculated. The mechanical fragility index (MFI) was determined by measuring the amount of Hb released in the supernatant of RBC rocked with stainless steel beads for 1 hour (h). Oxidative hemolysis levels were evaluated following treatment of RBC with 17 mM phenylhydrazine (PHZ) for 1 h at 37°C. Reactive oxygen species (ROS) and calcium accumulation were measured by fluorometry; the extracellular antioxidant activity and lipid peroxidation were determined spectrophotometrically; phosphatidylserine (PS) exposure and RBC membrane protein carbonylation were estimated by multicolor flow cytometry and western blotting, respectively (see the Online Supplementary Methods for details).