Nutridoma cs

Short tandem repeat authenticated HL-60 cells Saha et al. (2023) (link) were maintained in R10 media at 5% CO2 and 37°C and at a concentration of 0.2–1 million/mL by passaging them every 2–3 days. 5 days prior to experiments, HL-60s were differentiated into a neutrophil-like state by taking an aliquot of cells in their culturing medium and supplementing with an equal volume of Nutridoma-CS (Roche #11363743001) and DMSO diluted in RPMI such that that the final concentrations were 0.2 million/mL HL-60 cells, 2% (v/v) Nutridoma-CS, 1.3% (v/v) DMSO, 5% (v/v) FBS in RPMI. After 5 days at 37°C/5% CO2, we observed robust expression of terminal differentiation markers like FPR1 as reported previously Rincón et al. (2018) (link).
Lenti-X HEK-293Ts (Takara) were used for lentivirus production and maintained at below 80% confluency in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum. These cells were also maintained at 5% CO2 and 37°C. All cell lines were routinely monitored for mycoplasma contamination using standard mycoplasma monitoring kits (Lonza).

Hybridomas secreting anti-flavivirus monoclonal antibodies were generated from the most responsive JEV VLP-vaccinated mice (JEV FRµNT50 > 120) following the standard protocol¹¹¹ with minor modifications^{112 (link)}. Briefly, JEV VLP-vaccinated mice were boosted intraperitoneally with either 4 µg of purified JEV VLP or mD2VLP emulsified in Freund’s incomplete adjuvant (Sigma-Aldrich, St. Louis, MO, USA) for three consecutive days. Two days after the last boost, the splenocytes were harvested and then individually fused with NSI/1-Ag4-1 myeloma cells using 50% polyethylene glycol (PEG) (PEG Hybri-Max™, Sigma-Aldrich, St. Louis, MO, USA). Fused cells were resuspended in RPMI supplemented with 20% FBS, hypoxanthine-aminopterin-thymidine medium, and hybridoma growth factor (Nutridoma™-CS, Roche Diagnostics, Indianapolis, IN, USA). Two weeks post-fusion, supernatants from the selected polyclonal colonies were individually screened for secretion of mAbs by IgG ELISA as described above using JEV, DENV-2, and ZIKV VLPs, and detected with an HRP-conjugated goat anti-mouse IgG. The positive clones (P/N values of ≥2) were subcloned by limiting dilution to isolate monoclonal cells and then further screened for VLP ELISA binding and flavivirus neutralization activities using unpurified supernatants.