Fungizone

The B16LS9 murine melanoma cell line was kindly provided by Hans E. Grossniklaus (Emory University School of Medicine, Atlanta, GA). B16LS9 cells were derived from hepatic metastases following intracameral injection of B16-F1 cutaneous melanoma cells in C57BL/6 mice [24 (link)]. Tumor cells were maintained in complete DMEM medium containing 10% FBS (HyClone, Logan UT), 100 U/ml of penicillin, 50 ng of streptomycin, 0.1% Fungizone (BioWhittaker, Walkersville, MD), 2.0 mM glutamine (BioWhittaker), 0.01 M HEPES buffer (BioWhittaker), and 0.5% 2-Mercaptoethanol (Sigma-Aldrich, St. Louis, MO).

The virus neutralization assay was conducted with the cH9/1 virus using a 3-day incubation period [30 ,31 (link)]. Briefly, cell culture plates (Flat-bottom 96-well Nunclone Delta surface, USA) were seeded with 1.5×10⁴ MDCK cells/well and incubated at 37°C overnight. Next, heat-inactivated plasma samples were diluted to 1:10 and 2-fold serially diluted before incubation with cH9/1N3 (100 TCID₅₀/50 µl) for 1h at 37°C. MDCK cells were washed with phosphate buffered saline (PBS), and plasma/virus dilutions were added and incubated for 1 h at 37°C. The mixture was removed, cells were washed with PBS, and 50 µl of serially diluted plasma plus 50 µl infection medium (DMEM medium containing 2.5µg/ml tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Worthington Biomedical, USA), PSA (100 IU/ml penicillin, 100mg/ml streptomycin and 0.25 μg fungizone; Lonza, Switzerland) and 0.14% bovine serum albumin (Sigma-Aldrich, USA) were added to each well before incubation at 37°C for 72 h. The virus neutralization titers were measured by haemagglutination assay using the supernatant (50 µl) and 50 µl of 0.7% human red blood cells and read after 30 min of incubation at room temperature. The highest dilution of plasma giving complete haemagglutination was read as the neutralizing antibody titer.