Fluromax 2

The permeability of polarized LLC-PK monolayers was determined by measuring the transepithelial passage of FITC-dextran with molecular masses of 4 kDa and 70 kDa (FD4 and FD70, respectively) (Sigma-Aldrich), as described previously (30 (link)). Briefly, polarized LLC-PK cells grown on Transwell PET membrane inserts (Corning-Costar) were treated with or without chemicals or infected with or without PSaV Cowden strain (MOI of 50) in the presence or absence of 200 μM GCDCA for 5, 30, or 90 min at 37°C. As a positive control, another set of cells was treated with EGTA (1.8 mM) for 10 min. Afterward, FD4 and FD70 (10 μg/ml) were added to the apical chamber of the Transwell insert, and the cells were incubated for 1 h at 37°C. After the incubation period, the media were collected from the apical and basolateral sides, and the concentration of FITC-dextran was measured using a fluorometer (FluroMax 2; Horiba, Kyoto, Japan) at an excitation wavelength of 492 nm and an emission wavelength of 520 nm.

Paracellular flux was determined as described previously (5 (link), 73 (link)). Briefly, confluent LLC-PK cells grown on transwell inserts were treated with MEM only and treated or not with 200 μM GCDCA for 1 h or infected or not with PSaV strain Cowden (MOI = 50) or PSaV VLPs (4 μg/ml) in the presence or absence of 200 μM GCDCA for 1 h. As a positive control, 1.8 mM calcium ion chelating agent, EGTA, was used to treat another set of monolayers for 10 min. Subsequently, 250 μl of the tracer solution (10 μg/ml 4-kDa or 70-kDa FITC-dextran; Sigma-Aldrich) was applied to the apical sides of the confluent LLC-PK cells. In the mock-treated and/or mock-inoculated group, only medium was added to each well. The samples were incubated for 1 h at 37°C, and media from the upper and lower chambers were collected and measured using a fluorometer (FluroMax 2; Horiba, Kyoto, Japan) at 492 nm (excitation) and 520 nm (emission).

The permeability of polarized MDCK monolayers was determined by measuring the transepithelial passage of fluorescein-isothiocyanate (FITC) dextran with a molecular mass of 4 and 70 kDa (FD4 and FD70, respectively; Sigma-Aldrich), as described previously^{7 (link),60 (link)–62 (link)}. Briefly, polarized MDCK cells grown on polyester membrane transwells were treated with or without chemicals or infected with or without RVA strains DS-1 or NCDV (MOI = 10) for 5, 60, or 120 min at 37 °C. Another set of monolayers was incubated with 1.8 mM EGTA for 10 min as a positive control. Afterwards, FD4 and FD70 (10 μg/ml) were added to the apical chamber of the transwell, and the cells were incubated for 1 h at 37 °C. After the incubation period, media were collected from the apical and basolateral sides, and the concentration of FITC dextran was determined by a fluorometer (Fluro Max 2; Horiba, Kyoto, Japan) at an excitation wavelength of 492 nm and emission wavelength of 520 nm.