Veroe6

VeroE6/TMPRSS2 cells (African green monkey kidney-derived cells expressing human TMPRSS2, purchased from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, JCRB1819, were maintained in DMEM containing 10% FBS and 1 mg/ml G418 at 37 °C in 5% CO₂. The virus was propagated in VeroE6/TMPRSS2 cells and the virus titer was determined by the 50% tissue culture infectious dose (TCID₅₀) method and expressed as TCID₅₀/ml^{38 (link)}. The viral strains used are SARS-CoV-2/JP/Hiroshima-46059T/2020 (B.1.1, D614G, EPI_ISL_6289932^{39 (link)}), SARS-CoV-2/JP/HiroC77/2021, (AY.29, Delta, EPI_ISL_6316561), and SARS-CoV-2/JP/FH-229/2021 (BA.1.1, Omicron, EPI_ISL_11505197).
The serially diluted antibody (50 µl) was mixed with 100 TCID₅₀/50 µl of the virus and reacted at 37 °C for 1 h, then inoculated into VeroE6/TMPRSS2 cells to determine the minimum inhibitory concentration (MIC). Alternatively, the infectivity of the reacted antibody-virus mixture was measured by inoculating to 8 wells of a 96-well plate and observing cytopathic effects (CPE) or by the plaque assay using 10% methylcellulose to determine 50% effective dose (ED₅₀) of the antibody. SARS-CoV-2 infection was performed in the BSL3 facility of Hiroshima University.

Vero E6 (JCRB9007), Huh-7 (JCRB0403), and THP-1 (JCRB0112) cells were obtained from the Japanese Collection of Research Bioresources Cell Bank. HEK293T cells (CRL-3216) were obtained from the American Type Culture Collection. Cells were maintained using Dulbecco’s Minimum Essential Medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), 100 unit/ml penicillin, and 100 µg/ml streptomycin at 37 °C with 5% CO₂. The ISE6 and BME/CTVM23 tick cell lines were provided by the CEH Institute of Virology and Environmental Microbiology (Oxford, UK) and the Tick Cell Biobank at the University of Liverpool, respectively. Use of ISE6 cells was authorized by the University of Minnesota, the original provider of the cell line. The ISE6 cells were cultured in L-15B medium supplemented with 10% FBS, 5% tryptose phosphate broth (TPB, Merk) and 0.1% bovine lipoprotein concentrate (MP Biomedicals) at 32 °C^{25 (link)}. The BME/CTVM23 cells were maintained in L-15 (Leibovitz) medium supplemented with 20% FBS, 10% TPB and 2 mM l-glutamine at 32 °C^{6 (link)}.

The human liver cancer cell line, HepG2, was provided by SBI Pharmaceuticals Co. Ltd. (Tokyo, Japan). VeroE6, a cultured cell line derived from kidney epithelial cells isolated from African green monkeys, was obtained from JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition, Japan. The cells were cultured in DMEM (Low) culture medium containing 10% foetal bovine serum (FBS) and 10% ABAM, at 37°C in a 5% CO₂ incubator. Experiments were performed at a cell density of 50%–80% confluence.