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. 2020 Jun 11;140(2):183–208. doi: 10.1007/s00401-020-02174-2

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© The Author(s) 2020

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — Bacterial transmigration and permeability upon S. pneumoniae infection in brain ECs. a The scheme depicts the in vitro model of the BBB for meningitis infection where bEnd5 cells were cultured in 24-well PET transwells (10⁵ cells/cm²) and infected with S. pneumoniae (D39 and TIGR4 strain; MOI 50) along with the addition of labeled dextrans to the apical chamber (5 μM each of 3 kD TMR, and 70 kD FITC dextran). b Media supernatant from infected and control cells from the insert and the bottom well at 4 h post-infection were collected and centrifuged for plating and culture on blood agar plate. The colony forming unit (CFU) assay indicates transmigration of approximately 1% of the bacteria added to the insert (N = 5, ***p < 0.001, 2-tailed paired t test compared to control). c Confocal microscopy analysis of transwell inserts to confirm the presence of bacteria indicated predominant localization of the bacteria (green) at the cell–cell borders with VE-cadherin (red) serving as EC junction marker (representative from N = 3 experiments; scale bar 10 μm). d Permeability analysis of bEnd5 cells 4 h post-infection performed with low (3 kD) and high molecular weight (70 kD) dextrans shows a significant increase in dextran permeability for both sizes, confirming the breakdown of endothelial barrier due to infection (N = 5, *p < 0.05, **p < 0.01, 2-tailed paired t-test compared to control). e Representative graph for continuous transendothelial electrical resistance (TEER) values of S. pneumoniae (D39) infected bEnd5 cells on inserts using the cellZscope® system setting the values of the control group between 3–4 h post-infection to 100%. The graph shows reduced TEER values starting 3 h post-infection that persists for several hours indicating increase in paracellular permeability. The increase in the TEER values of the control group from the plateau at 0 h reflects the changes due to electrode handling and media composition. f Quantification at 4 h shows a significant reduction in TEER values in infected cells compared to control (N = 4, *p < 0.05, 2-tailed paired t test)