Epithelial volt ohm meter

Confluent monolayers of NCM460 cells or isolated primary colonic epithelial cells were grown in 24-well Transwell chambers (polycarbonate membrane, filter pore size 0.4 μm, area 0.33 cm²; Costar) for 24 h, and then TER was measured at 37 °C using an Epithelial Volt Ohm Meter (Millipore, Billerica, MA, USA). TER values were calculated by subtracting the blank filter and by multiplying the surface area of the filter. All the measurements were performed in triplicate.

A549 monolayer grown in Transwell filter units (3-μm-diameter pores, Corning, New York, NY, USA) were left uninfected or infected with 1 MOI of H5N1 virus and then incubated in the absence or presence of the indicated inhibitors. The monolayers were analyzed for transepithelial resistance (Epithelial Volt-‘Ohm Meter, Millipore, St. Louis, MO) at the indicated time after virus infection. The measured values were calculated by multiplying the measured electrical resistance by the area of the filter. The results represent the mean ± SD of three independent experiments.

The primary HAE-ALI cultures HAE-ALI^B3-20, HAE-ALI^B4-20, and HAE-ALI^B9-20 were provided by the Cells and Tissue Core of the Center for Gene Therapy, University of Iowa (18 (link), 55 (link)– (link)57 (link)). These polarized HAE-ALI cultures were derived from three independent donors. The freshly isolated human bronchial epithelial cells from the donor tissues were seeded onto collagen-coated, semipermeable polycarbonate membrane inserts (0.33 cm², 0.4-μm pore size, Costar Transwell, catalog no. 3413, Corning, New York), and grown at an ALI as previously described (58 (link)). The cultures were maintained in USG medium containing 2% Ultroser G (USG) serum substitute (Pall BioSepra, France). After 3 to 4 weeks of culture at an ALI, the polarized culture was fully differentiated. The polarity of the HAE was determined for the TEER using an epithelial volt-ohm meter (Millipore). A value of 1,000 Ω·cm² or higher was chosen for SARS-CoV-2 infection as we previously used for HBoV1 infection (50 (link), 59 (link)). HAE-ALI^L209 cultures on 1.1 cm² Millicell-PCF (Millipore, Billerica, MA) were provided by Dr. Matthias Salathe, which were generated following a published method (60 (link)) using primary airway bronchial epithelial cells isolated from the lung of a donor (L209).