Figure 2.

Real-time fluorescence imaging of Rhodamine Red Tfn endocytosis. (A) Time lapse imaging. Cells cultured on glass coverslips were either exposed to Rhodamine Red holo-Tfn, lissamine rhodamine apo-Tfn, or first treated with CPZ followed by washout with plain MEM/BSA and then treated with Rhodamine Red holo-Tfn. Confocal imaging of live cells was carried out simultaneously in the green and red channels. The measurements started ∼30 s after cell exposure to the ligand. Representative images taken at different measurement times (indicated in s at each frame) in a specific focal plane, where the red fluorescence was maximal, are shown. The images, which were selected from the respective Movie S1, Movie S2, and Movie S3, were processed by contrast enhancement, using ImageJ software. Scale bar = 5 μm. (B) Quantitative analysis. The intracellular fluorescence intensity of Tfn recorded from at least five different cells in each case was averaged and normalized to the extracellular background. The intracellular accumulation of holo and apo-Tfn in melanoma cells, and of holo-Tfn in CPZ-treated cells is shown. (C) Representative image. The image shows how an area of interest (solid white line), basically defined by SRG delineating the cell boundaries at a particular optical section, was chosen for the fluorescence quantitative analyses of Tfn internalization. Scale bar = 5 μm.