Fig 1.
Transendothelial electrical resistance (TEER) and tracer passage.
TEER expressed in ohm.cm2 (A). Permeability of FITC-dextran and sodium fluorescein (B) across the bEnd.3 and ihBMEC monolayers grown on Transwell inserts. Reduction in the spontaneous passage of FITC-dextran (C) and sodium fluorescein (D) by the bEnd.3 and ihBMEC monolayers. Spontaneous migration represents the transport of the tracer in the absence of the brain endothelial monolayer. Data are presented as mean ± SEM of n = 3–8 per time point. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, by two-way ANOVA and Holm-Sidak post hoc test.
Fig 2.
Correlation between TEER measurements and tracer permeability across the bEnd.3 and ihBMEC monolayer.
A significant inverse correlation was observed between FITC-dextran and TEER using the bEnd.3 cells (Pearson r = -0.77, p<0.0001) (A), and between sodium fluorescein and TEER while using the ihBMECs (Pearson r = -0.73, p<0.01) (B).
Fig 3.
Endothelial phenotypic confirmation of bEnd.3 cells and ihBMECs.
Phase-contrast images showing the typical cobblestone morphology of the bEnd.3 and ihBMECs (A, scale bar = 250 μm). Uptake of AlexaFluor-488 labeled acetylated low-density lipoprotein (acLDL) by the bEnd.3 cells and ihBMECs (shown as green in B). Immunofluorescent detection of Von Willebrand factor (vWF) (shown as green in C) and claudin-5 (shown as green in D) in bEnd.3 cells and ihBMECs. The nucleus is stained with DRAQ5 (shown as red). Scale bar = 40 μm. Data are presented as mean ± SEM of n = 3 independent repeats. *p<0.05 by Student’s t-test.
Fig 4.
Vascular phenotype, response to inflammatory stimuli, and erythrophagocytic phenotype of ihBMECs.
CD31 immunostaining in the ihBMECs (A) and the bEnd.3 cells (B). Tube-like formation by the ihBMECs grown on collagen and fibronectin coated Transwell inserts (C). ihBMECs respond to two inflammatory stimuli, LPS and TNFα, and show a significant reduction in TEER measurements (D). H&E-stained images show robust attachment and engulfment of tBHP-treated red blood cells with ihBMECs (E). Aged red blood cell internalization was further confirmed by maximum projection and orthogonal confocal microscopy images of H&E-stained cells showing the ihBMECs and tBHP red blood cells in autofluorescent green signal (F). Arrowheads show aged red blood cells internalized by the ihBMECs in E and F. Scale bar = 40 μm in A-B, 100 μm in C, 50 μm in E, and 20 μm or 10 μm in F. Data are presented as mean ± SEM of n = 3 independent repeats, and *p<0.05, **p<0.01 by two-way ANOVA and Holm-Sidak post hoc test in D.