Ligation of CR1 increases RBC membrane deformability and depends on extracellular Ca⁺⁺. (A) CR1-mediated increase in membrane deformability depends on Ca⁺⁺. Freshly isolated RBCs were incubated with either control Ab or anti-CR1 mAb in the presence of Ca⁺⁺ and Mg⁺⁺ (Ca⁺⁺) or Mg⁺⁺-EGTA (EGTA) for 10 minutes at RT. RBCs were washed, and 30 cells were analyzed for each condition by LOT. The recoil time (t_c) of CR1-ligated RBCs (60 ± 2 milliseconds) was significantly shorter (P < .001) than the recoil time of control RBCs (72 ± 3 milliseconds). The bars represent 95% confidence interval. (B) Ligation of RBC CR1 decreases the time required for RBC to pass through microchannels. Sequential images (3 frames/s) of a RBC entering the microfluidic device are shown in the top panel. The extent of the decrease in the time required for RBCs to pass through the microchannels depends directly on the number of CR1 molecules on the surface of RBCs. Each dot in the graph represents a RBC. These results are representative of 5 independent experiments using different anti-CR1 Abs and different high and low CR1 donors.