Figure 1.
Transepithelial electric resistance (TEER) of Caco-2 monolayers before and after 120 minutes permeability experiments.
Cell layers were treated with 0.05-RAMEB (FR) alone or in the presence of 5 mM RAMEB (FRR). Untreated monolayers were kept in HBSS. Values are expressed as means ± SD, n = 9 for FR, n = 6 for FRR treatment and n = 6 for untreated samples. There were no significant differences between TEER values before and after the treatments (p>0.05) and among the groups (p>0.05).
Figure 2.
Accumulation of FITC-RAMEB in Caco-2 monolayers after 120 minutes permeability experiments.
Caco-2 monolayers were treated with 0.05 mM FITC-RAMEB (FR) alone or in combination with 5 mM RAMEB (FRR) for 120 minutes. Monolayers were washed and the fluorescence intensity of the accumulated FITC-RAMEB was determined with FLUOstar Optima microplate reader. Presented values are means ± SD, n = 7 for FR, n = 4 for FRR treatment and n = 5 for untreated samples. FR and FRR treatments increased significantly the fluorescence of monolayers compared to the untreated control (p<0.001).
Figure 3.
Kinetics of FITC-RAMEB uptake in Caco-2 monolayers.
Cell monolayers were treated with 0.05-RAMEB (FR) alone or in combination with 5 mM RAMEB (FRR) and in different time points the incubation was stopped. After washing cells were fixed with 3% paraformaldehyde solution and the accumulated FITC-RAMEB was determined by microplate reader. Cell nuclei were labeled with DAPI and fluorescence intensities of FITC-RAMEB were normalized for DAPI fluorescence intensities. Values are expressed as means ± SD, n = 3 for FR and FRR treatments.
Figure 4.
Release of FITC-RAMEB from Caco-2 monolayers.
Treatment with 0.5-RAMEB was carried out on Transwell® inserts for 120 minutes, then the monolayers were washed and FITC-RAMEB release was followed in the apical and basolateral chambers during the next 120 minutes. FITC fluorescence intensities were determined in the control group of the samples after the first 120 minutes and considered as 100% accumulation. The rate of release in the second group was compared to this value. Values are means ± SD, n = 4.
Figure 5.
Confocal images of undifferentiated Caco-2 cells.
Cells were treated with the solution of 0.05-RAMEB and 5 mM RAMEB (FRR). FITC-RAMEB (green) is localized in small vesicles (white arrows) under the CellMask labeled cell membrane (red) or in larger vesicles near the DAPI stained cell nucleus (light blue). Aggregated particles of FITC-RAMEB can be also seen outside the cell membrane (A). Nine consecutive confocal sections of a cluster of cells were recorded (B). Each section is one and half micrometer thick. FITC-RAMEB (green) is located in cytoplasmic granules. The granular bright particles are observed inside the cell membrane and outside of the cell nuclei.
Figure 6.
Cyclodextrin enters differentiated Caco-2 cells of a high resistance Caco-2 cell layer.
A confluent layer was treated with 0.05-RAMEB and imaged by confocal microscope in twelve two-micrometer thick sections, of which six are demonstrated in the panels on the left side (A–F). On the right, one middle section of the image shows the top view of the cell layer (H) at the level indicated by blue lines in side sections. Upper (G) and right (I) side images are appropriate sections from perpendicular directions at green and at red lines. Crosshair (green and red lines at the long white arrow) set to an intense FITC-RAMEB (green) granule (indicated by arrows), which is located at the nuclear (light blue DAPI stain) level of cells. Several smaller FITC-RAMEB green granules can be seen below the cell membrane marked by CellMask (dark blue).
Figure 7.
FITC-RAMEB colocalized with Rab5 proteins.
RFP-Rab5a transfected Caco-2 cells were treated with 0.05 mM FITC-RAMEB (green) for 30 minutes. Colocalization is indicated by yellow pixels in the confocal microscopic images on the sections marked by the 18-micrometer and 21-micrometer label. Figure shows nine subsequent, three-micrometer thick confocal sections giving altogether a twenty-four-micrometer cross layer of a Caco-2 cell. CellMask (dark blue) was used for labeling membrane at the cell surface and nucleus was stained by DAPI (light blue). Rab5 proteins (red) are visualized by transient transfection of a plasmid coding Red Fluorescent Protein (RFP) tagged Rab5.
Figure 8.
Cellular uptake of calcein-AM (A), FITC-RAMEB (B) and Lucifer Yellow (C) as a function of ligand concentration.
Cells were treated at 37°C and 0°C and the cellular fluorescence was determined by flow cytometry, after excluding dead cells with propidium iodide.(Graphs show results of a representative experiment).
Figure 9.
Effect of 10 µM rottlerin on the cellular uptake of FITC-RAMEB and Lucifer Yellow.
Caco-2 cells were pre-incubated for 45 minutes with rottlerin and the internalization of the fluorescent molecules was detected by flow cytometer. (n = 3, p<0.01).