University of Pretoria
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Cell permeability and tight junction analysis using multiple imaging and molecular techniques

posted on 2024-06-12, 08:49 authored by Jaime de CarvalhoJaime de Carvalho

The dataset contains all the raw quantitative data and microscopy images generated for a research project aiming to develop and validate a contact coculture model of the blood brain barrier using bEnd.3 and C8-D1A cell lines. Growth curves were generated using and transforming the raw data generated from the crystal violet assay for the bEnd.3 and C8-D1A cell lines using a range of seeding densities. In addition, phase-contrast micrographs were taken at 24 hour intervals throughout the 96 hour culture period to confirm the growth and confluence of the cell cultures. This was used to assess the growth kinetics of the different seeding densities for both cell lines to ensure the cells were viable throughout the cell culture period and to select optimal seeding densities for further experimentation.

Thereafter, a paracellular permeability assay was conducted using bEnd.3 cells, over a range of seeding densities, cultured for 48, 72, and 96 hours. The bEnd.3 cells were then treated with 5 mM ethylene glycol-bis(β-aminoethyl ether)-tetraacetic acid (EGTA) for 3 and 24 hours in order to validate the formation of functional endothelial barriers. This generated raw data regarding the permeability of the bEnd.3 cell line and used to obtain permeability values. Similarly, paracellular permeability data was generated for the bEnd.3/C8-D1A contact coculture model using the paracellular permeability assay. Raw data on the permeability of the bEnd.3/C8-D1A contact coculture was generated and used to obtain apparent permeability values.

The 10 000 cells/well seeding density for bEnd.3 cells was used to assess the dynamics of tight junction protein (i.e. claudin-5, occludin, and zona occludens 1) localisation over time using confocal microscopy. Further confocal micrographs were taken at 96 hours of the 10 000 cells/well bEnd.3 cells: a. untreated; b. treated with 5 mM EGTA for 3 hours and; c. following 30 minutes of calcium restoration. The C8-D1A cells were subjected to reverse transcriptase polymerase chain reaction to validate the expression of aquaporin 4 (AQP4), glutamate aspartate transporter (GLAST), glutamate transporter 1 (GLT-1), glial fibrillary acidic protein (GFAP). Genomic DNA, no transcript controls, and a housekeeping gene was included to confirm the results of the assay.




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