BBB barrier function was evaluated by measuring TEER. The resistance values (Ω) were measured using a Millicell ERS-2 (Merk Millipore). The TEER values were calculated as previously described [10 (link), 11 (link)]. We confirmed the value of TEER was peaked on day 10. Therefore, all experiments were performed on day 10.
The cells were washed with Dulbecco’s phosphate-buffered saline (DPBS, Sigma) supplemented with D-glucose and HEPES (DPBS-H). DPBS-H containing 10 μg/mL sodium fluorescein (NaF, Sigma) as a paracellular marker was then added into the upper chamber for 30 min at 37 °C. After incubation, the medium in the lower chamber was collected and then the concentration of NaF in the medium was measured using a fluorescence multi-well plate reader (Genios, TECAN).
The cells were washed with Dulbecco’s phosphate-buffered saline (DPBS, Sigma) supplemented with D-glucose and HEPES (DPBS-H). DPBS-H containing 10 μg/mL sodium fluorescein (NaF, Sigma) as a paracellular marker was then added into the upper chamber for 30 min at 37 °C. After incubation, the medium in the lower chamber was collected and then the concentration of NaF in the medium was measured using a fluorescence multi-well plate reader (Genios, TECAN).
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