Figure 1. Prolonged refrigerated storage of murine RBCs leads to increased erythrophagocytosis in vitro.

Erythrophagocytosis assays were performed at 37°C in 5% CO₂, in complete medium, for 1 hour, at a RBC:macrophage ratio of 50:1. Results are presented as mean ± s.d. (A) J774A.1 cells were incubated with fresh RBCs (“Fresh”) or RBCs stored at 4°C for 14 days (“Old”). Following osmotic shock to remove non-ingested RBCs, cells were photographed with a phase-contrast microscope (original magnification: 400×). Arrows indicate RBCs ingested by J774A.1 cells. (B) J774A.1 cells were incubated with PBS, fresh RBCs, or 14-day refrigerator-stored (i.e., old) RBCs. The amounts of ingested RBCs were quantified by harvesting cells following osmotic shock, lysing the resulting J774A.1 cells, and measuring hemoglobin content. (C) J774A.1 cells were incubated with PBS or with RBCs stored at 4°C for 2, 8, 17, or 24 days. The amounts of ingested RBCs were quantified, as in Panel B. (D) J774A.1 cells were incubated with fresh RBCs or 14-day refrigerator-stored RBCs in the presence or absence of LPS (100 ng/mL). The amounts of ingested RBCs were quantified, as in Panel B. (E) Primary cultures of mouse splenocytes were incubated with PBS, fresh RBCs, or 14-day refrigerator-stored RBCs. The amounts of ingested RBCs were quantified, as described in Panel B. Shown are representative examples of at least 3 experiments.