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. 2020 Jul 3;11:712. doi: 10.3389/fphys.2020.00712

FIGURE 1.

FIGURE 1

During storage in K+/EDTA tubes at 4°C, RBCs rapidly lose biconcavity and energy, become more fragile and form surface vesicles. RBCs stored for 0 to 4 weeks (w) in K+/EDTA tubes at 4°C were isolated and analyzed for morphology (A,B), size (C), fragility (D), and intracellular ATP content (E). The time needed to reach 50% effect and the corresponding statistical analysis (Mann–Whitney U test) are indicated above the vertical dotted line. (A) Scanning electron microscopy images of RBCs on filters. RBCs were fixed in increasingly concentrated glutaraldehyde solutions, deposited on filters, post-fixed with osmium tetroxide, covered with gold and observed by electron microscopy. Representative images of 1 experiment including 3 preparations per storage time. (B,C) Abundance and surface area of spherocytes vs. discocytes. RBCs were immobilized on poly-L-lysine (PLL)-coated coverslips, imaged and classified into two categories, the discocytes (disco; circles) and the spherocytes (sphero; squares), which are smaller and possess higher circularity. In (B), data are means ± SEM of 7 independent experiments where at least 100 RBCs were analyzed. In (C), data are means ± SEM of 4 independent experiments where at least 150 RBCs were analyzed. (D) RBC osmotic fragility. RBCs were incubated at RT in media of decreasing osmolarity. After centrifugation, the hemoglobin (Hb) content in the supernatant and in the pellet were measured by spectrophotometry at 450 nm. The horizontal dotted line indicates the medium osmolarity at which 50% of the RBCs were lysed. Data are means ± SD of 1 experiment representative of 2. (E) Intracellular ATP content. RBCs were lysed and incubated with firefly luciferase and luciferin. The intensity of the light emitted following the oxidation of luciferin to oxyluciferin by luciferase in the presence of ATP was then measured by luminescence. The intracellular ATP concentration was normalized on the Hb content and then expressed in % of fresh RBCs (i.e., 0 weeks of storage). Data are means ± SEM of 4 independent experiments. *p < 0.05; **p < 0.01; ****p < 0.0001.