Fig. S8.

A4 treatment does not induce an anti-A4 antibody response and sufficiently saturates cell surface CD47. (A) High binding plates were coated with an irrelevant Nb (Nb2, Left) or A4 (Right) at 1 μg/mL in carbonate buffer overnight, washed, and blocked with 10% inactivated fetal calf serum (IFCS) before incubation with serial dilutions of mouse serum from untreated mice (naive) or mice treated with daily doses of Nb control or A4 for 2 wk. Bound mouse antibody was detected using an anti-IgG conjugated to HRP (Southern Biotech). (B) Mice were injected i.p. with 200 μg of A4 or Nb control on days 0 and 1. Twenty-four hours after the second dose, spleen cells were harvested from each mouse and incubated for 15 min with anti-mouse CD47 conjugated to PE (miap301; BD Biosciences). PE fluorescence was determined in RBCs from Nb control-treated mice, A4-treated mice, and spleen cells preincubated with excess A4 (ex vivo block) by flow cytometry. RBCs were identified using forward scatter (FSC)/side scatter (SSC) gating. (C) Spleen cells from mice treated as in B were incubated with anti-CD47 PE and stained with antibodies to CD3, CD4, CD8, CD11b, and CD19 to identify lymphoid and myeloid populations. The percentage of CD47 bound to A4 was determined by calculating the difference between the average maximum PE signal in Nb control-treated mice compared with mice treated with A4 [(control MFI – A4 MFI)/control MFI]. MFI, mean fluorescent intensity. Values were normalized using the fluorescence from spleen cells treated ex vivo with excess A4.