IgG antibodies against band 3 and GPA rigidify RBCs, whereas IgGs against RhCE and RhD do not. Representative ektacytometric curves (at least 3 separate donors studied per antibody) of RBCs treated with antibodies targeting (A) RhD or RhCE, (B) band 3 or Wr^b, or (C) GPA. A 5% suspension of RhD⁺ human RBCs in PBS was treated with 100 nM of the indicated antibody clones (∼100 000 IgG per RBC). After incubation for 1 hour at 37°C, the red cell suspensions were read on an ektacytometer in 5.5% polyvinylpyrrolidone. Figure keys indicate antibody clones. (D) Ektacytometric dose response of anti-RhCE vs anti-Wr^b IgG antibodies. Selected antibody clones against RhCE (BRIC69, red) and Wr^b (BRIC14, blue) were added at 100 nM to varying hematocrit RBC suspensions (5%, 10%, and 20%, 6 donors tested) to result in ligand ratios of 25 000 to 100 000 IgG per RBC. aWr^b demonstrated a significant increase in SS1/2 at all ligand-loading ratios, whereas no significant difference was seen for aRhCE antibodies. Mean ± SD is shown, n = 3-6 for each condition. (E) Flow cytometry on aRhCE (BRIC69, red) and aWr^b (BRIC14, blue) IgG-treated RBCs stained with Alexa Fluor 488–labeled anti-mouse secondary antibodies shows no significant difference in bound IgGs (based on median fluorescence) at the indicated loading ratios. (F) Representative histogram demonstrating similar antibody loading for RBCs treated with aRhCE (BRIC69, red) and aWr^b (BRIC14, blue) antibodies.