Figure 3.

Effect of media composition on RBEC barrier formation and characteristics. (a) Comparison of the effects of the DMEM/MVGS and EBM-2/EGM-2 media formulations on the TEER of RBECs grown for 14 days on cell culture inserts. Data is presented as mean ± SEM and was analysed using an unpaired, two-tailed students t-test, ***P < 0.0001; n = 5 independent cell culture experiments in 24-well plates, with 3 inserts per experiment, equivalent to 15 inserts total . (b) Calculated permeability coefficients for the paracellular passage of 100 μM (50 μg/mL) Lucifer yellow over a 90 minute period at 37°C across RBEC monolayers on cell culture inserts cultured in DMEM/MVGS and EBM-2/EGM-2 media formulations. Data is presented as mean ± SEM and was analysed using an unpaired, two-tailed students t-test, ***P < 0.0001; n = 5 independent cell culture experiments, with 3 inserts per experiment, equivalent to 15 inserts total. Fluorescence microscope images of RBECs stained with an antibody raised against the tight junction protein claudin-5 following culture in (c) DMEM/MVGS supplement, or (d) EBM-2/EGM-2. White arrows indicate regions of discontinuous claudin-5 staining. Images are representative of 3 independent cultures, with five fields of view taken from each individual preparation of cells using the 20× objective on an Olympus IX81 microscope. (e) Western blot analysis of claudin-5 protein expression levels in RBECs cultured in DMEM/MVGS and EBM-2/EGM-2. Blots were reprobed with anti-actin antibodies as a control for equal loading of cell lysates. (f) Densitometry analysis of claudin-5 band intensity, normalised to actin levels, for RBECs grown in DMEM/MVGS vs. EBM-2/EGM-2. Data is presented as mean ± SEM and was analysed using an unpaired, two-tailed students t-test, *P < 0.01; n = 3 independent experiments.