Gentamicin sulfate

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.

Dulbecco's Modified Eagle Medium (DMEM) with and without phenol red, 0.05% trypsin/0.53 mM EDTA and L-glutamine were all purchased from Gibco (Grand Island, NY). Fetal bovine serum (FBS) was purchased from Atlanta Biologicals (Lawrenceville, GA). Gentamicin Sulfate was from Corning (Corning, NY). TransIT-X2TM transfection reagent was purchased from Mirus (Madison, WI). FuGENE 6 and FuGENE HD Transfection Reagents were purchased from Roche Diagnostics (Indianapolis, IN). All restriction enzymes, DNA polymerase I (Klenow) and High Efficiency Competent E. Coli Cells [NEB 10-beta; Cat. No: C3019H] were from New England BioLabs (Ipswich, MA). Hi-Lo DNA Markers were from Minnesota Molecular, Inc. (Cat. No: 1010, Minneapolis, MN). Dimethyl Sulphoxide (DMSO) and tetramethylbenzidine were purchased from Sigma-Aldrich (St. Louis, MO). DNeasy Blood and Tissue and RNeasy Mini kits were from Qiagen (Germantown, MD). USB VeriQuest Fast SYBR Green qPCR Master Mix with Fluorescein Kit was from Afflymetrix (Santa Clara, CA). SuperSignal West Dura was purchased from Pierce (Rockford, IL). Enzyme-free Cell Dissociation Buffer, was from ThermoFisher Scientific (Waltham, Massachusetts). NucBlue Live Cell Stain (Cat. No: R37605) was from Invitrogen (Carlsbad, CA).

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.