Sodium bicarbonate 7.5 solution

293T cells were transfected with pcDNA3.1 plasmid vectors encoding F or F2A VLPs constructs using Transporter 5 transfection reagent (Polysciences Europe GmbH) according to the manufacturer’s protocol. Transfection was performed at 37°C for 72 h and 96 h posttransfection. Next, samples were collected from the cell culture medium, and VLPs were precipitated using a solution of 10% polyethylene glycol 6000 (PEG6000) supplemented with 300 NaCl overnight at 4°C. Proteins were pelleted and dissolved in 50 mM Tris/100 mM NaCl/50 mM EDTA buffer. The protein solutions were further used to analyze prM/M and E protein expression.
F2A VLPs for further experiments were produced using an optimized protocol. 16 h posttransfection, the cells were supplemented with 2 mM sodium butyrate, 1× MEM Non-Essential Amino Acids Solution (Gibco), and 0.075% of Sodium Bicarbonate 7.5% solution (Gibco) and were further cultured at 28°C or 37°C. Medium from transfected cells was harvested 96 h posttransfection and clarified via centrifugation at 3,500 × g for 10 min at 4°C followed by filtration through a 0.45 μm PVDF filter. The supernatant containing VLPs was stored at 4°C until further analysis or purification.

DMEM (1×) Glutamax-I, DPBS, penicillin-streptomycin (P/S), fetal bovine serum (FBS), 1× 0.05% trypsin-EDTA (T/E), B-27, HEPES (1 M), and sodium bicarbonate 7.5% solution were purchased from Gibco, Thermo Fisher Scientific (Waltham, USA). EA and D-luciferin were purchased from Cayman Chemical, Ann Arbor, USA, and PanReac AppliChem, Darmstadt, Germany, respectively. DMEM low-glucose powder, D-(+)-glucose, UA, dexamethasone (Dex), and dimethyl-sulfoxide (DMSO) were purchased from Sigma Aldrich, St. Louis, USA. Acridine Orange Propidium Iodide (AO-PI) was purchased from Logos Biosystems, Gyeonggi-do, South Korea.

MDCK cells were seeded in a sterile 96-well cell culture plate at a density of 25,000 cells/well using complete DMEM. The next day, cells were infected with a multiplicity of infection (MOI) of 3.0 for 16h using serum free minimal essential media (MEM, Gibco), supplemented with penicillin/streptomycin, HEPES (Gibco), glutamine (200mM 1-glutamine, Gibco) and sodium bicarbonate (sodium bicarbonate 7.5% solution, Gibco). The cells were fixed with 3.7% paraformaldehyde (PFA)/PBS for 1h at RT. The plate was the blocked for 1h with 3%milk/PBS. The antibodies were diluted to 30 µg/ml in 1% milk/PBS and incubated for 1h at RT. An anti-Lassa antibody [(KL-AV-1A12 (53 (link))] was included as a negative control and CR9114 (56 (link)), a broadly cross-reactive HA antibody, as a positive control. The plate was washed three times with 1xPBS and then incubated with a fluorescence goat anti-mouse IgG heavy plus light chain (H+L)–Alexa Fluor 488 antibody (Abcam), which was diluted 1:1000 in 1% milk/PBS. The cells were washed with PBS and kept in PBS to avoid drying out during microscopy (Olympus IX-70).