Increased lipid peroxidation and oxidative stress in Gpx4^Δ erythroid cells does not impair their life span in the periphery. (A) Measurement of lipid peroxidation in unchallenged peripheral TER119⁺ erythrocytes using the lipophilic redox-sensitive dye BODIPY 581/591, which upon oxidation, shifts its fluorescence from red to green. Data are mean ± SE; n ≥ 7. (B) ROS in unchallenged peripheral TER119⁺ erythrocytes using the redox-sensitive dye 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂-DCFDA) (n ≥ 7). (C) Survival of biotin-labeled peripheral TER119⁺ erythrocytes. Data are mean ± SE; n ≥ 6. (D) Increased number of peripheral CD71⁺/TER119⁺ reticulocytes in Gpx4^Δ mice within the first 4 weeks after poly(I:C) administration. Data are mean ± SE; n ≥ 5. (E-F) Representative images of TUNEL assay in spleen sections and 4,6 diamidino-2-phenylindole (DAPI) as a counterstain showing increased cell death in Gpx4^Δ mice. Image acquisition was performed using a Zeiss Axio Imager M2 with 40×/0.95 korr Apochromat objective (magnification ×400). Flow cytometry analysis using PI to determine the number of nonviable CD71⁺ cells in bone marrow (G) and in spleen (H). Data are mean ± SE; n ≥ 3. (I) Formation of o-dianisidine–positive erythroid colonies from bone marrow in methylcellulose semisolid media. Red colony formation of Gpx4^Δ bone marrow cells could be rescued by addition of α-tocopherol to the medium. Data are mean ± SE; n ≥ 3. *P < .05, **P < .01, ***P < .001 by Student t test. α-Toc, α-tocopherol; n.s., not significant; PI, propidium iodide.