Figure 4.

Time course of transgene expression in vitro

231/LM2-4-EGFPLuc cells were transfected (A and B) in T-75 flasks with psCV1-hIgG1 or (C and D) in T-25 flasks with psCV1-hIgG1 or pmCherry or mock transfection (buffer only) for 4 h in basal medium, and thereafter the same amount of complete medium was added. Cells were harvested and analyzed at (A and B) the indicated time point or (C and D) 48 h after transfection (p0) and passaged further (ratio 1:8) on day 5 (p1), day 9 (p2), and day 12 (p3) into new T-25 flasks with fresh complete medium, and remaining cells were analyzed by flow cytometry. (A and C) Cells stained with anti-CD47 antibody [B6H12.2] or IgG isotype (secondary antibody goat anti-mouse IgG H&L Alexa Fluor 647). (B and D) Cells stained with goat anti-human IgG Fc-DyLight 650 or goat IgG isotype AF647 conjugate. n = 6 +SD. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001; U test (Mann-Whitney).