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Fig. 8. Internalization kinetics of the I/F8/RIP immunotoxin. A2774 cells were incubated with either I/F8/RIP (large and small left-hand panels) or RIP alone (small right-hand panels) at 4°C in the presence of the anti-RIP mAb CY12.14 as dimerizing agent. Cells were then left on ice (A,B) or transferred to 37°C for different times to induce internalization (C,D,E), and were subsequently untreated (small panels) or acid-washed to strip the surface-bound molecules (large panels). Samples were then fixed, permeabilized, stained by FITC-labeled secondary antibody and nuclei were counterstained with Propidium Iodide. Samples were visualized by using an Olympus confocal laser microscope system. Crosslinking the immunotoxin with anti-RIP mAb allowed the visualization of the internalized fraction at concentrations as low as 1 x10^–8 M I/F8/RIP (about 0.2 µg/ml). This amount of immunotoxin was used, as it is the highest concentration that still shows a 100-fold difference in toxicity when compared to free RIP. Original magnification, 600 x.

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