Figure 2.

CD47-blockage and sCV1-hIgG1 binding after in vitro transfection

231/LM2-4 cells were transfected in T-75 flasks with 40 μg of indicated plasmid for 4 h in basal medium (thereafter same amount of complete medium added), harvested after 48 h, and analyzed by flow cytometry. CD47 blockage was evaluated by staining with anti-CD47 antibody clone B6H12.2 competing with sCV1-hIgG1 for CD47 binding and isotype IgG staining as control (secondary antibody goat anti-mouse IgG H&L Alexa Fluor 647). sCV1-hIgG1-binding to the cell surface was detected by goat anti-human IgG Fc-DyLight 650 staining or goat IgG isotype-AF647 conjugate. (A) Histogram plots. Left: CD47 stain. Right: IgG-Fc stain. (B) Average signal intensity (geometric mean of individual measurements). Top: CD47 staining. Bottom: IgG-Fc staining. n = 12 + standard deviation (SD). ∗∗∗∗p ≤ 0.0001, ns p > 0.05; U test (Mann-Whitney).