Millicell ers electrode

Cells were maintained in DMEM with 10% FBS, 1× nonessential amino acids, and 1× penicillin and streptomycin at 37°C with 5% CO₂. Cells of passage 35-40 were used in this study to maintain relatively constant cellular phenotypes. The medium was replaced every 2-3 days during incubation. After reaching approximately 80% confluence, the cells were detached using 0.02% ethylenediaminetetraacetic acid (EDTA) and 0.05% trypsin at a density of 5.3×10⁴ cells/cm² on a 60-mm plastic culture dish for the uptake experiment or a 12-mm polyester membrane insert in a 12-well plate for the permeation experiment. The media in the culture plates were changed every two days for the first week after seeding and replaced daily afterward. The integrity of the cell monolayers was examined by measuring the transepithelial electrical resistance (TEER) with a Millicell-ERS-electrode (Millipore Corp, Billerica, MA, USA). After 21-23 days, monolayers with a TEER value (TEER=[TEER_test- TEER_background]×area) above 500Ωcm² were used during the transport experiments.

Barrier integrity or monolayer tightness was assessed by measuring transendothelial electrical resistance (TEER) while using a Millicell ERS electrode (Millipore, Bedford, MA, USA). Paracellular permeability was assessed in monolayers by measuring the diffusion profile of 0.1 µCi of [¹⁴C] sucrose (PerkinElmer, Waltham, MA, USA). Radioactivities of samples were counted using a liquid scintillation counter (PerkinElmer, Waltham, MA, USA). The permeability coefficient was calculated while using following formula [34 (link)]: $PC=\frac{dQ}{dt}\times \frac{1}{A\times C_0}$
where dQ/dt is rate of [¹⁴C] sucrose diffusion, A is area of insert, and C₀ is the initial concentration of [¹⁴C] Sucrose added in donor compartment.