Ligation of RBC CR1 promotes Ca⁺⁺ influx that depends on the genetically determined CR1 levels. (A) Antibody ligation of RBC CR1 triggers a Ca⁺⁺ influx. Intra-RBC Ca⁺⁺ concentration of Fluo-4–loaded RBCs from known high and low CR1 expressors was measured by flow cytometric analysis for 20 seconds before RBC CR1 was ligated (arrow) by anti-CR1 mAb. Changes in intra-RBC Ca⁺⁺ concentration were measured for a total of 2 minutes. (B) Ligation of CR1 by complement opsonized-particles promotes RBC Ca influx. Intra-RBC Ca⁺⁺ concentration of Fluo-4–loaded RBCs from known high and low CR1 expressors was measured by flow cytometry for 20 seconds before RBC CR1 was ligated (arrow) by control or complement opsonized beads. Each line represents the average RBC fluorescence analyzed at a rate of approximately 1000 RBC/second. (C) Characterization of RBC CR1 levels by antibody shows large gap between low and high CR1 expressors. RBCs from known low and high CR1 expressors were incubated with anti-CR1 mAb followed by AlexaFluor488 secondary Ab and analyzed by flow cytometry. (D) Functional characterization of RBC CR1 using complement-opsonized beads identifies a narrower gap between RBCs from high and low expressors. RBCs from known low and high CR1 expressors were incubated with fluorescently labeled complement opsonized beads for 30 minutes, washed, and analyzed by flow cytometry. (E) Fluorescence microscopy overlapped with phase contrast microscopy of RBCs from a high CR1 expressor after incubation with fluorescently labeled complement opsonized beads. (F) Fluorescence microscopy overlapped with phase contrast microscopy of RBCs from a low CR1 expressor after incubation with fluorescently labeled complement opsonized beads. Bar represents 10 μm. The results shown here are from the same individuals and are representative of 3 independent experiments using 3 different high CR1 and 2 different low CR1 expressors.