Millicell ha filters

MDCK cells (CCL-34 and _frt transfectants) were grown on 12 mm Millicell-HA filters with a pore size of 0.45 μm (Millipore, Billerica, MA). 10⁵ cells were seeded on each filter and subsequently cultured in 24-well trays for 2–4 days to obtain a confluent monolayer. Tightness of the cell layer was evaluated each day with a modified fluorescein leakage test [46 (link)] where 10 μg/L sodium-fluorescein (Sigma-Aldrich) was added to the apical side of cells for 15 min. at 37°C before measuring the degree of leakage into the basolateral media fraction using an ELISA reader. The cell layer was considered tight when the leakage was less than 5% compared to a control filter without cells. Cells grown on 0.4 μm PET 12 well hanging culture inserts (Millipore) were evaluated by trans-epithelial electrical resistance measurements. Briefly, filters were placed in an Endohm-12 culture cup (World Precision Instruments, Sarasota, FL), and resistance was measured in triplicates according to manufacturers instructions using an EVOM voltohmmeter (World Precision Instruments). Cells with background-substracted resistances above 100 Ω were considered tight.