Mammarenaviral Infection Is Dependent on Directional Exposure to and Release from Polarized Intestinal Epithelia

Warner, Nikole L.; Jokinen, Jenny D.; Beier, Juliane I.; Sokoloski, Kevin J.; Lukashevich, Igor S.

Caco-2 cells were seeded on 24-well plate Transwell inserts (Corning, New York, NY, USA) with a 0.45 micron filter as previously described [36, 37] . Briefly, 160,000 cells in 0.5 mL were plated on "apical" side of insert, and 1 mL of nutrient media was added to each well. For basolateral seeding of the Caco-2 cells, the Transwell inserts were flipped upside down in a sterile container, and 0.5 × 10 6 cells in a volume of 100 µL was placed on each Transwell surface. The cells were incubated for 1 h at 37 • C to allow for adherence to the membrane. Cells were then put back into the 24-well plate and 0.5 mL of nutrient media was added to each insert. Cells were maintained at 37 • C in 5% CO 2 for 21 days until polarization was completed. Media was changed every 2-3 days with fresh media for the entirety of the polarization process. To determine if polarization of epithelial monolayers was complete, Transepithelial Electrical Resistance (TEER) was measured with an EVOM2 Epithelial Voltohmmeter (World Precision Instruments, Sarasota, FL, USA) as previously described [38] . In experiments with non-polarized Caco-2, cells confluent monolayers on day 3 after seeding were used. Tight junction proteins were analyzed with western blot (WB) and qRT-PCR. Antibodies for WB were obtained from the following: ZO-1 (61-7300), Claudin-1 (Thermo Scientific 51-9000, Waltham, MA, USA), Occludin (Thermo Scientific 71-1500). Pre-made primers/probe sets against human ZO-1 (Hs01551861) and Occludin (Hs00170162) were purchased from ThermoFisher.