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. 2021 Jun 10;9:668296. doi: 10.3389/fcell.2021.668296

FIGURE 1.

FIGURE 1

Primary mouse brain endothelial cell characterization at passage 0. (A) Following isolation, brain endothelial cells establish a confluent monolayer. Capacitance (dashed lines) measured at 64 kHz reflects cell attachment and spreading and decreases as cell coverage increases; capacitance plateaus below 10 nF (black dotted line) once a monolayer is established (ECIS® ZΘApplied Biosystems). APOE3 and APOE4 brain endothelial cell cultures establish a monolayer by 48 h, with no genotype differences. At confluence, there are no differences in (B) cell density (n = 28) or (C) BrdU+ nuclei (<5%) between APOE3 and APOE4 brain endothelial cells. (D) APOE3 and APOE4 brain endothelial cells express the tight junction protein markers claudin-5 (green) and occludin (red), and exhibit classical endothelial cell morphology (narrow, elongated, tightly packed cells) when assessed by immunocytochemistry, scale bar = 20 μm. (E) When impedance is measured at lower frequencies (i.e., 4000, 2000, and 1000 Hz), more current passes between the cells and is therefore a measure of paracellular permeability referred to as transendothelial electrical resistance (TEER). At all frequencies, TEER (solid lines in A) progressively increases over the course of 48 h and is then maintained for at least 4 days for both APOE genotypes. The frequency that represents paracellular permeability to the greatest extent is cell type-dependent and can be estimated empirically (Stolwijk et al., 2015). 1000 Hz represents the optimal frequency, as the ratio of cell-covered electrode:cell-free electrode is higher (∼22) compared to 2000 Hz (∼20) and 4000 Hz (14) for both APOE3 and APOE4 brain endothelial cells. At 1000 Hz TEER values are lower in APOE4 brain endothelial cells compared to APOE3. Data is expressed as mean ± S.E.M. *p < 0.05 by Student’s t-test with n = 6 (unless otherwise specified above).