Lucifer yellow

TER was measured using Cellzscope (nanoAnalytics, Münster, Germany). MDCK-II cells were seeded on hanging PET cell culture inserts (Merck Millipore, #PIHT12R48) at a density of 80,000 cells/insert. After TER values had reached a stable plateau, the cells were transferred to a 24-well plate and washed twice with HBSS+/+ warmed to 37°C. Lucifer yellow (m.w. 457 Da; Sigma-Aldrich, #L0259) was dissolved in warm HBSS+/+ (200 μM) and 200 μl solution were applied to the apical compartment of the insert, in order to determine the permeability coefficient of the cell barrier [23 (link)]. The basolateral compartment was filled with 1ml warm HBSS+/+ and the plate was incubated at 37% with 10% CO₂ for 10 min. The inserts were then moved to a fresh 24-well plate containing prewarmed HBSS+/+ and incubated for two more 10 min periods. From each basolateral compartment, 80 μl of sample were transferred to a 96-well plate in triplicate and Lucifer yellow fluorescence was measured using a Tecan microplate reader (Tecan, Männedorf, Switzerland); concentrations were determined from a standard curve.

At the end of RE preparation, REs were held at room temperature without culture plate covers for 1 hour to remove any residual humidity. Then, trans-epidermal water loss (TEWL) was measured on Biox Aquaflux (Biox Systems Ltd, Bishop's Stortford, UK) according to the manufacturer's instructions. Three independent technical replicates were performed for each RE (3 REs per condition).
At the end of preparation, 200 μl of 1 mM Lucifer yellow (Sigma, St-Louis, MO, USA) was added on the RE surface. After incubation at 37°C for 6 hours, the Lucifer yellow concentration in the culture medium was measured by fluorescence in a microplate reader (Infinite M1000, Tecan, Männedorf, Switzerland) with excitation at 425 nm and emission at 550 nm. Three independent technical replicates were performed for each RE (3 REs per condition).