Figure 3

Ad-IFNγ induced apoptosis in NPC cells. Both floating and adherent NPC cells were harvested after infection with 50 MOI of Ad-IFNγ or Ad-LacZ for 72 hr, washed with ice-cold PBS and followed by apoptosis analysis. (A) Annexin V-FITC/7-AAD binding assay by flow cytometry. 5×10⁵ of collected cells were incubated in the dark for 15 minutes with 100 μL of 1×Binding Buffer containing 10 μL of Annexin V-FITC and 20 μL of 7-ADD Viability Dye (Beckman-Coulter, Inc. Marseille, France) on ice. Samples were diluted with 400 μL of 1×Binding Buffer and immediately analyzed by a Coulter Epics Altra flow cytometer (Beckman-Coulter). Left, Diagrams in a representative experiment. Annexin V⁺/7-AAD⁻ indicates early apoptotic cells, and Annexin V⁺/7-AAD⁺ indicates late apoptotic cells. Right, Statistical analysis of Annexin V-positive cells (apoptotic cells) generated from three independent experiments. (B) Hoechst 33342/PI double staining. Collected cells were adjusted to the density of 1×10⁶ cells/mL in PBS with 1% FBS and stained with 5 μM of Hoechst 33342 at 37°C for 10 min. And then cells were stained with 1 μM of PI for 10 min at room temperature after washing with PBS to remove Hoechst dye. The stained cells were mounted onto a polylysine-coated slide and examined under a fluorescent microscope. A total of 300~400 nuclei from 5~8 randomly chosen fields were examined. High blue fluorescent indicates apoptotic cells (bright blue arrow), low blue indicates live cells (azury arrow), while red represents dead cells (pink arrow). Apoptosis was expressed as a percentage of the total number of nuclei examined. Left, Representative pictures from one experiment. Right, Statistical analysis of apoptotic cells from three independent experiments. ** p<0.01, compared with Ad-LacZ-treated cells.