Figure 6.

Immunocytochemistry of brain endothelial junctional space following growth in either Enriched Media or Minimal Media. Cells were seeded at time 0 h at 20,000 cells per 0.3 cm². (A) ECIS resistance and R_b measurements over 120 h. Cells were grown in either Enriched Media or Minimal Media from T = 0 h, with media changed at T = 48 h (I). Data shown is the mean ± SD (n = 3 wells) of one independent experiment representative of three experimental repeats; (B) Cell number count of Hoechst stained nuclei at time point II, obtained through Image J Software analysis. Data shown is the mean ± SEM (n = 18 wells) of 1 independent experiment representative of 3 experimental repeats; (C) The junctional space following growth in either Enriched Media or Minimal Media 72 h post seeding. The time point of fixation corresponds to the second vertical dotted line (II) shown on the ECIS traces. Representative GFP/DAPI and GFP monochrome images for the junctional proteins CD144, ZO-1, β-catenin, and α-catenin are shown. Each panel shows cells grown in Minimal Media on the left and cells grown in Enriched Media on the right. The corresponding Alexa Fluor 488 (GFP) monochrome image is shown below each merged image. Green—junctional protein, blue—nuclei. Scale bar is 200 µm. Immunocytochemistry data show one representative image from one independent experiment, which is representative of three experimental repeats. Graphical representations of p values are * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.