Mitochondrial membrane potential and ROS production during red cell maturation. Mitochondrial membrane potential (ΔΨm) was monitored using MitoTracker CMXRos as described in “Mitochondria membrane potential measurement.” (A) on mature erythrocytes obtained from a healthy rat (dotted line) and immature red cells obtained from an anemic rat (solid line). (Inset) Mature erythrocytes (1 μL packed cell volume), young reticulocytes (1 μL packed cell volume) corresponding to the lower-density fraction of Percoll-purified immature red cells,¹⁴  and mitochondria (50-μg protein) isolated from reticulocytes were loaded on 10% SDS-PAGE and analyzed by Western blotting for 15-lipoxygenase (15-LOX). The 15-LOX (1.4-μg protein) isolated from rabbit reticulocytes was loaded as a control. The molecular mass (kDa) standards are indicated on the left. (B) The ΔΨm was monitored after 6, 20, 30, and 45 hours of in vitro maturation of Percoll-purified young reticulocytes (as indicated). (C) The ΔΨm was monitored on freshly isolated young reticulocytes (tinted pattern) and after 48 hours of cell maturation in the absence (dotted line) or presence (solid line) of the lipoxygenase inhibitor ETYA (30 μM). (Inset) Reticulocytes were matured in vitro for 24 hours in the absence (CTRL) or presence of the lipoxygenase inhibitors (NDGA, ETYA) at the indicated concentrations. Exosomes were then collected from the culture medium, loaded on 10% SDS-PAGE, and analyzed for the presence of TfR by Western blot. (D) Intracellular ROS was assayed using the dye hydroethydine (HE) on mature (–) and immature red cells (—).