The kinetics of the distribution of apo-Tf–Texas Red and Fe-Tf–Bodipy in Caco-2 cells.

Multiple Caco-2 cell monolayers were incubated either with 0.1 μm apo-Tf–Texas Red (panels A and B) or 0.1 μm Fe-Tf–Bodipy (panels C and D) and were examined by confocal microscopy after 7, 14, and 21 minutes of incubation (panels A and C). After 21 minutes, the basal chamber contents were changed so that the cells containing apo-Tf–Texas Red were now offered Fe-Tf–Bodipy (panel B) in the basal chamber, and the cells that had seen Fe-Tf–Bodipy were now offered apo-Tf–Texas Red (panel D). The cells were again examined after an additional 7, 14, and 21 minutes (panels B and D). At the indicated times, the cells were then washed and processed, and the nuclei were stained blue with ToPro-3. The graphs on the y-axis show the average intensity of the fluorescent transferrins obtained by image analysis of 2 to 4 independent experiments for each treatment with duplicate monolayers. The fluorescence intensity for each of the 4 panels was normalized to the highest intensity occurring at the 3 times. The x-axis is the distance in arbitrary units from the membrane on which the cells were grown. In each monolayer, 10 to 23 optical slices were analyzed. The cell monolayers averaged 251 cells per monolayer. The blue bar indicates the area encompassed by the nuclei.