FIG. 2.

Internalization rates of carboxyfluorescein-Ad5, -Ad7, and -Ad5f7. Carboxyfluorescein-Ad (10¹¹ particles/ml) was incubated with A549 cells at 4°C to evaluate only surface binding or at 37°C to allow binding and internalization of Ad. At each time point, cells were washed, fixed, and treated with HRP and DAB, with or without H₂O₂. Extracellular fluorophore was quenched by precipitation of DAB by HRP in the presence of H₂O₂. (A) A549 cells incubated with carboxyfluorescein-Ad5 (4°C for 1 h) and treated with HRP-DAB without H₂O₂. (B) Same as panel A, but treated with HRP-DAB in the presence of H₂O₂. (C) A549 cells infected with carboxyfluorescein-Ad5 at 37°C for 10 min, washed, and incubated for an additional 10 min at 37°C, then treated with HRP-DAB without H₂O₂. (D) Same as panel C, but treated with HRP-DAB in the presence of H₂O₂. (E) A549 cells incubated with carboxyfluorescein-Ad7 (4°C for 1 h) and treated with HRP-DAB without H₂O₂. (F) Same as panel E, but treated with HRP-DAB in the presence of H₂O₂. (G) A549 cells infected with carboxyfluorescein-Ad7 at 37°C for 10 min, washed, and incubated for an additional 10 min at 37°C, then treated with HRP-DAB without H₂O₂. (H) Same as panel G, but treated with HRP-DAB in the presence of H₂O₂. (I) A549 cells incubated with carboxyfluorescein-Ad5f7 (4°C for 1 h) and treated with HRP-DAB without H₂O₂. (J) Same as panel I, but treated with HRP-DAB in the presence of H₂O₂. (K) A549 cells infected with carboxyfluorescein-Ad5f7 at 37°C for 10 min, washed, and incubated for an additional 10 min at 37°C, then treated with HRP-DAB without H₂O₂. (L) Same as panel K, but treated with HRP-DAB in the presence of H₂O₂. Bar = 10 μm. (M) Percentage of internalized Ad per cell relative to the total amount of Ad per cell. Levels of internalized Ad5 (●), Ad7 (○), and Ad5f7 (▵) were determined by digital image analysis (see Materials and Methods). Data for zero internalization time correspond to surface-labeled cells (4°C binding for 1 h).