Human SIRPα 1ex binding to RBCs is CD47 specific and species specific. Human RBC (A) or Ig-CD47-coated beads (B) were incubated with soluble SIRPα1ex or GST, and bound protein was detected in flow cytometry using fluorescent anti-GST. SIRPα1ex specifically binds to human RBCs whereas GST does not. Blocking antibodies (B6H12 and BRIC126) inhibit the CD47-SIRPα interaction. 2D3, a nonblocking antibody, enhances SIRPα1ex binding to RBCs but not Ig-CD47 beads, probably by ability to cluster CD47 (C). Using the same methodology, RBCs from 5 mammalian species were labeled with SIRPα1ex or GST (D) under standardized conditions of cell number and reagent concentration. Human SIRPα1ex binds to human RBCs as expected and does not bind to RBCs from cow, mouse, or rat. Human SIRPα1ex also binds to pig RBCs and results in higher intensity in comparison to human RBCs. Note that ^* indicates a slight signal above background.