Nanogel Permeability Across bEnd3 Monolayer

Murine brain capillary endothelial cells bEnd3 have been grown until confluency on the semi-permeable membrane of Transwell inserts (24-well plate) treated with Matrigel according to the protocol (Simon 2010 (link)). Trans-endothelial electrical resistance (TEER) was measured on Day 5 using E-VOM2 Epithelial Voltmeter (World Precision Instruments, Sarasota, FL). Confluent monolayers had resistance values ≥33 Ωcm². Upper Transwell compartment (apical side) medium containing 10% FBS was mixed with stock nanogel solution. The lower Transwell compartment (basolateral side) contained fresh serum-free medium. Plates were incubated for 0.5, 1, 2 and 4 h at 37°C, when fluorescence of basolateral solution was measured at ex 590 nm/em 620 nm in black-walled plate in duplicates. Results have been analyzed and P_app calculated using the following equation: P_app = (dx/dt)/(0.33 × C_o × 60 × 60) (cm/s), where x is the nanogel accumulation on the basolateral side during 1h-incubation, C_o is the initial nanogel concentration on the apical side, and 0.33 is the filter area in cm².