Maintenance mem medium

All experiments using infectious EBOV were performed under the Biosafety Level 4 (BSL-4) containment of the Galveston National Laboratory. Vero-E6 cell monolayers were grown in 12 or 24-well plates and treated for 1 hr at 37°C, 5% CO₂ with compounds diluted in maintenance MEM medium (Life Technologies) containing 2% FBS (Hyclone, Logan, UT), 0.1% gentamicin sulfate (Corning, Corning, NY), 1% Nonessential Amino Acids (Sigma), and 1% sodium pyruvate (Sigma). Cell monolayers were infected with the recombinant EBOV that expresses eGFP (EBOV-eGFP) from an added gene for 1 hr at 37°C [23 (link)]. After adsorption, monolayers were washed three times with phosphate buffered saline (PBS), a fresh compound in the maintenance medium was added to each well, and monolayers were incubated at 37°C for 4 days (passage 1), 11 days (passages 2 and 3) or 10 days (passage 4) post-infection.
To quantify EBOV-eGFP titers in Vero-E6 cell supernatants, confluent Vero-E6 monolayers were inoculated with 10-fold serially diluted supernatants, adsorbed for 1 hour at 37°C, 5% CO₂ and subsequently coated with an overlay of the medium containing 0.9% methylcellulose (Sigma, St. Louis, MO). Fluorescent viral plaques were counted after 4 days using a UV microscope.

Vero-E6 cell monolayers were grown in 24 well plates and treated with compounds diluted in maintenance MEM medium (Life Technologies, Grand Island, NY, United States) containing 2% FBS (Hyclone, Logan, UT), 0.1% gentamicin sulfate (Corning, Manassas, VA, United States), 1% Non-essential Amino Acids (Sigma, St. Louis, MO, United States), and 1% sodium pyruvate (Sigma) for 1 h at 37°C, 5% CO₂. Cell monolayers were infected with the recombinant EBOV that expresses eGFP (EBOV-eGFP) from an added gene (Hoenen et al., 2013 (link)) for 3 h at 37°C. Following adsorption, monolayers were washed three times with PBS, fresh compound in the maintenance medium was added to each well, and monolayers were incubated for 48 h at 37°C. To determine titers of EBOV-eGFP, supernatants were titrated on Vero-E6 cell monolayers covered with an overlay of the medium containing 0.9% methylcellulose (Sigma). After 4 day-long incubation at 37°C, fluorescent viral plaques were counted under a UV microscope. Experiments with EBOV-eGFP were performed in the BSL-4 facilities of the Galveston National Laboratory, UTMB.