Matrigel coated 12 well transwell inserts

This assay was performed using the model developed by Cecchelli and co-workers. ^{68 (link)} Endothelial cells and pericytes were defrosted in gelatin-coated Petri dishes (Corning). Pericytes and endothelial cells were cultured in DMEM pH 6.8 or in supplemented endothelial cell growth medium (Sciencells), respectively. After 48 h, pericytes (50000 cells/well) and endothelial cells (80000 cells/well) were seeded in gelatin-coated 12-well plates or in Matrigel-coated 12-well Transwell inserts (Corning), respectively. Medium was changed every 2–3 days, and assays were performed 7–8 days after seeding. Lucifer Yellow (50 µM) was used as internal control (P_app <15 × 10⁻⁶ cm/s), and LY fluorescence was measured in a 96-well plate with a Fluoroskan Ascent microplate fluorometer (Thermo Fisher Scientific).
Compounds were dissolved in Ringer Hepes at the final concentration of 200 µM. Then, 500 µL of the compound and 1500 µL of Ringer HEPES alone were introduced in the apical or in the basolateral compartments, respectively. The plates were set on at 37 °C for 2 h. The solutions from both compartments were then recovered and quantified by UPLC and identified by UPLC-MS and MALDI-TOF. Apparent permeability was calculated using eq 2:
$$P_{\text{app}}=\frac{\mathrm{d}{Q}_{\mathrm{A}}(t)}{\mathrm{d}t}\frac{1}{A}\frac{1}{Q_{\mathrm{D}}(t_0)}{V}_{\mathrm{D}}$$