Transwell filter plate

To establish polarized pBCEC cultures (Kober et al., 2017 (link)), cells were trypsinized and plated onto collagen‐coated (120 μg ml⁻¹) 12‐well transwell filter plates (0.4‐μm pore size, Corning) at a density of 40,000 cells cm^‐2. Cells were grown for 2–3 days depending on the transendothelial electrical resistance (TEER; 50 Ω cm⁻²). The tightness of the transwell culture was assessed by measuring TEER using an EndOhm tissue resistance measurement chamber and EndOhm ohmmeter (World Precision Instruments, Florida). TEER of collagen‐coated, cell‐free filters were used as blanks. Tight junction formation was induced (overnight) by adding DMEM/Ham's F‐12 medium containing 550 nM hydrocortisone, 1% penicillin/streptomycin and 0.7 mM l‐glutamine. Establishment of intact tight junctions was indicated by TEER rising above 150 Ω cm⁻². Agents known to increase BBB permeability, 27OH (0.055 and 0.5 μM), 24OH (0.5 μM), 7β‐hydroxycholesterol (7βOH; 0.5 μM) or LPS (5 μM), were used as positive controls and were added to apical media containing 1% BSA. TEER were measured at indicated time points for up to 24 h. Six independent experiments with the above additions and use of aliquots from the same cell culture were performed (Figure 5). In addition, two separate independent experiments with other cell cultures were performed with n = 3.

Calu-3 cells were gifts from Dr. Wing-Hung Ko at The Chinese University of Hong Kong, Hong Kong, China. The cells were cultured with DMEM/F12 supplemented with 10% fetal bovine serum, 1% MEM NEAA, and 100 U/mL penicillin-streptomycin solution at 37 °C in a humidified atmosphere of 5% (v/v) CO₂. The cultured medium was changed every 2–3 days and the cells were passaged weekly before seeding. To obtain confluent cell monolayers, Calu-3 cells were seeded on Transwell^® filter plate (12 mm diameter inserts, 0.4 μm pore size, Corning, NY, USA) at a density of 5 × 10⁵ cells/well with 500 μL medium in the apical chamber and 1500 μL medium in the basolateral chamber. After 2 days, the apical medium was removed to create an air-liquid interface culture, and the medium of basolateral chamber was changed to 1000 μL. The medium was changed every 2 days.