Penicillin streptomycin mixture

Virus infections were performed on Vero ATCC CCL-81 or SH-SY5Y ATCC CRL-2266 cells (ATCC, Manassas, VA). Vero cells were cultured in minimal essential medium (MEM) (Corning, Manassas, VA) supplemented with 10% fetal bovine serum (Gibco, Life Technologies, Paisley, UK), and SH-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)–F-12 medium (Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum. A penicillin-streptomycin mixture (Corning) and l-glutamine (Corning) were also added to cell cultures. WNV New York 99 (60 (link)), USUV SAAR 1776 (60 (link)), American ZIKV PA259459 (61 (link)), a cell-passaged derivative of DENV-2 16681 (16 (link)), VSV Indiana (61 (link)), and CVB5 Faulkner (62 (link)) were used. Procedures for infections in liquid medium and virus titrations in Vero cells in semisolid agar medium were previously described (62 (link), 63 (link)). WNV, USUV, ZIKV, VSV, and CVB5 titers were determined at 24 h postinfection (p.i.). DENV-2 titers were determined at 48 h p.i. A multiplicity of infection (MOI) of 1 PFU/cell was used for all experiments unless otherwise indicated.

All animal experiments were conducted in accord with the permission of the Local Bioethics Committee. Primary cortical neurons were isolated from Wistar rat embryos at embryonic day 18.5 (E18.5). Pregnant females were euthanized in a CO₂ chamber, and the embryos were immediately removed in order to microdissect the brains. The cortices were excised, transferred to Hank’s Balanced Salt Solution (HBSS, Gibco, Waltham, MA, USA), supplemented with a 1% penicillin/streptomycin mixture (Gibco, Waltham, MA, USA) and washed. Afterwards, the tissue was trypsinized (2.5% trypsin, Gibco, Waltham, MA, USA) for 10 min at 37 °C, and homogenized gently by re-suspension with a sterile tip of a pipette. The cell suspension was centrifuged in 300 rcf for 4 min and re-suspended in Neurobasal-A medium (Gibco, Waltham, MA, USA) supplemented to final concentration with 1 mM sodium pyruvate, 12.5 mM glucose 2% B27 (Gibco, Waltham, MA, USA), 200 mM L-glutamine, 10 mM glutamic acid, a 1% penicillin/streptomycin mixture, and seeded in a 24-well plate poly-D-lysine (PDL)-coated (BioCoat, Corning, Tewksburry, MA, USA) in the number of 200,000 cells per well. The neural cultures were incubated in a CO₂ chamber (5%, 95% air balance, 98% humidity, 37 °C) for 14 days and were used on culture day in vitro (DIV) 14–15.