Figure 3.

Bystander effect and fusion protein secretion in vitro

231/LM2-4 cells were transfected for 48 h, and the supernatant was harvested thereafter and either analyzed for fusion protein content or transferred onto untransfected 231/LM2-4 cells. (A) Cells treated with transfection supernatant for 1 h (diluted with complete medium at the indicated ratio) and thereafter stained with anti-CD47 antibody [B6H12.2] (secondary antibody goat anti-mouse IgG H&L Alexa Fluor 647, left) or protein A (Alexa Fluor 488 conjugated, right). Representative histogram blots shown. Red line, cells incubated with supernatant from psCV1-hIgG1-transfected cells; blue line, cells incubated with supernatant from mock-transfected (buffer only) cells. (B) Dot blot analysis (2 μL/dot) with anti-human IgG Fc HRP antibody. Top (dots in duplicates): 48-h transfection supernatant diluted with complete medium at the indicated ratio, BSA solution (2 mg/mL), complete medium. Bottom: indicated amounts per dot of human IgG1 isotype. (C) Cells treated with 48-h transfection supernatant for 1 h (right) or supernatant from mock-transfected (buffer only) cells (left), washed, fixed, and stained with DAPI (blue) and goat anti-human IgG Fc (DyLight 650). Confocal laser scanning microscopy (CLSM) imaging was conducted with a 40× oil objective; representative middle sections of the cells are shown. Scale bars: 20 μm.