Cell permeability and tight junction analysis using multiple imaging and molecular techniques

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.

该数据集包含了为开发与验证基于bEnd.3和C8-D1A细胞系构建的血脑屏障接触共培养模型而生成的一切原始定量数据和显微镜图像。通过将晶紫染色法所获得的bEnd.3和C8-D1A细胞系的原始数据转换为生长曲线，并利用不同接种密度进行了处理。此外，在96小时的培养周期内，每隔24小时进行相位对比显微镜图像的采集，以证实细胞培养的生长和汇合。此过程用于评估两种细胞系不同接种密度的生长动力学，以确保细胞在整个培养周期内的活性，并选择最佳接种密度以进行后续实验。随后，在48、72和96小时的文化培养中，使用bEnd.3细胞在一系列接种密度下进行了细胞旁路通透性实验。随后，bEnd.3细胞被用5 mM乙二醇双(β-氨基乙醚)-四乙酸（EGTA）处理3小时和24小时，以验证功能性内皮屏障的形成。这生成了关于bEnd.3细胞系通透性的原始数据，并据此获得了通透性值。类似地，利用细胞旁路通透性实验生成了bEnd.3/C8-D1A接触共培养模型的细胞旁路通透性数据，并据此获得了表观通透性值。使用10,000个细胞/孔的接种密度对bEnd.3细胞进行了研究，以利用共聚焦显微镜评估紧密连接蛋白（即闭锁蛋白-5、紧密连接蛋白和封闭蛋白1）随时间的变化动态。在10,000个细胞/孔的bEnd.3细胞培养96小时后，进一步采集了以下共聚焦显微镜图像：a.未经处理；b.用5 mM EGTA处理3小时；c.钙离子恢复30分钟后。C8-D1A细胞接受了逆转录聚合酶链反应，以验证水通道蛋白4（AQP4）、谷氨酸天冬氨酸转运蛋白（GLAST）、谷氨酸转运蛋白1（GLT-1）和胶质纤维酸性蛋白（GFAP）的表达。为了确认实验结果，实验中还包括了基因组DNA、无转录本对照以及管家基因。