Journal: Cancer biology & therapy

Article Title: Cancer gene therapy of adenovirus-mediated anti-4-1BB scFv in immunocompetent mice.

doi: 10.4161/cbt.7.3.5425

Figure Lengend Snippet: Figure 2. Characterization of anti-4-1BB scFv expression. (A) RT-PCR analy- sis of anti-4-1BB scFv mRNA expression. Cultured cells were infected with Ad.CMV.AP or Ad.4-1BB scFv at a MOI of 10 for 24 h. The transcripts of anti- 4-1BB scFv were semi-quantified by RT-PCR with β-actin as an internal control. (B) Flow cytometric analysis of anti-4-1BB scFv cell surface expression. Hepa 1-6 cells were infected with Ad.4-1BB scFv or Ad.CMV.AP at a MOI of 10 for 24 h and stained with FITC-conjugated anti-human IgG F(c) antibody that recognized the immunoglobulin Fc fragments expressed on Ad.4-1BB scFv infected Hepa1-6 cells for flow cytometry analysis. (C) Visualizing anti-4-1BB scFv expression on the surface of Hepa1-6 cells by direct immunofluorescence staining. Cells were infected with Ad.4-1BB scFv or Ad.CMV.AP at a MOI of 30 for 24 h, fixed, incubated with FITC-conjugated anti-human IgG F(c) anti- body, and counterstained with Hoechst 33342. The same optical field was photographed under fluorescence light and normal light. The blue fluores- cence corresponded to cell nuclei, and the green fluorescence corresponded to anti-4-1BB scFv-hIgG Fc. Original magnification, x630.

Article Snippet: After 24 h of infection, cells were harvested, washed twice with ice-cold PBS and incubated in a 1:250 dilution of a fluorescein isothiocyanate (FITC)-labeled goat anti-human IgG F(c) (Rockland Immunochemicals Inc., PA, USA) on ice for 1 h, which recognized the immunoglobulin Fc fragments expressed on virus infected Hepa1-6 cells.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Infection, Control, Staining, Flow Cytometry, Immunofluorescence, Incubation, Fluorescence