Figure 1. hCMEC/D3 cells form a confluent monolayer following 10 days in culture.

(A) hCMEC/D3 cells were seeded on porous PTFE transwell inserts (0.4 μM) at time zero and cultured for 10 days. Throughout this time course, monolayer growth was visualized under 20X Relief Contrast Microscopy using an Olympus IX81 de-convolution microscope. (B) hCMEC/D3 cells remained in culture 2, 4, 6, or 10 days (as in A) prior to addition of 2 mg/mL 70 kDa FITC-D to the upper chamber. Samples were taken from the lower chamber at 5 minute intervals over 30 minutes in order to calculate Pe. (C) hCMEC/D3 cells were cultured (as in A) for 10 days prior to treatment of the monolayer with mannitol (1.4 M) for 30 minutes at 37°C in CO₂ (5%). Following treatment, a FITC-D assay was performed (as in B) to calculate Pe. Bars represent the mean of three independent experiments performed in triplicate with standard deviation. Statistical analysis was performed with a two-tailed Student’s t-test. *p < 0.0001.