Transfusions of old RBCs induce oxidative stress, PTGS2 expression, and cell death in RPMs. (A) Wild-type C57BL/6 mice were transfused with 350 μL of fresh or old RBCs and sacrificed at defined time points. Splenic RPM cell numbers were quantified posttransfusion. Old RBC transfusions induced a substantial decrease in RPMs by 2 hours posttransfusion, the levels of which returned to baseline 2 days later. (B) The gating strategy first selects for splenic nucleated non-RPM cells, which thereby selects for GFP⁺ (Csfr1/CD115⁺) cells; this is followed by gating out B cells, granulocytes, DCs, monocytes, and RPMs. The remaining cells were quantified to determine changes following transfusions of old, as compared with fresh, RBCs. (C) MaFIA mice were transfused with 350 μL of fresh or old RBCs and sacrificed 5 hours posttransfusion. Total non-RPM–nucleated cell numbers in the spleen were quantified, showing no differences. (D) Wild-type C57BL/6 mice were infused with PBS or supernatant from 350 μL of an old RBC blood bank and RPM cell number was quantified 5 hours posttransfusion. (E) Annexin V staining of RPMs following transfusion of old and fresh RBCs. (F) Upregulation of PTGS2 mRNA expression in RPMs after old RBC transfusions. Increases in ROS production (G) and lipid peroxidation (H) in RPMs following transfusions of old RBCs. *P < .05; **P < .01; ***P < .001; ****P < .0001; ANOVA with the Sidak multiple comparisons test. PTGS2 gene expression analysis was analyzed for significance using the unpaired Student t test. Data are representative of 3 independent experiments. FITC, fluorescein isothiocyanate.