Rpmi 1640 medium

HEK293T cells were grown in RPMI 1640 medium (Wisent, #350-035-CL) supplemented with 10% FBS (Wisent, #080-150) and 1% Gluta-Plus (Wisent, #609-066-EL). Cells were seeded in 15 cm tissue culture plates (Sarstedt, #83.3903) at a density of 7 × 10⁶ cells per plate one day before transfection. On the day of transfection, cells were co-transfected with lentiviral plasmid vectors, psPAX2 (Addgene, #12259), and pCMV-VSVG (Cell biolabs, #320023) using the jetPRIME transfection reagent (Polyplus, #CA89129-924) according to manufacturer’s protocol. Supernatant containing viral particles was collected at 48- and 72-hours post transfection and filtered through a 0.45 µm PVDF membrane (Sigma, #SE1M003M00). Viral particles were precipitated in 40% (w/v) polyethylene glycol (Sigma, #89510-1KG-F) overnight. On the next day, the viral particles were collected by centrifugation at 3,248 g for 30 mins at 4 °C. The pellet was resuspended in HBSS (Gibco, #14170112) + 25 mM HEPES (Thermo Fisher, #15630-080) and stored at −80 °C for long term storage.
For lentiviral transductions, non-TC-treated 24 well plates were coated with 20 µg/mL of Retronectin (Takara, Cat # T100B) for 2 hours at room temperature followed by aspiration and blocking with PBS containing 2% (w/v) BSA (Wisent Bioproducts, Cat # 800-096-EG) for 30 mins at room temperature. After aspiration of the blocking buffer, the concentrated virus suspension was added to wells. The plates were then centrifuged at 3248 g for two hours at 4 °C to allow virus binding. Following centrifugation, unbound virus was aspirated, and cells were added. The plates were then transferred to a 37 °C incubator to initiate lentiviral infection.

Whole-cell ELISAs were performed with a panel of representative strains of N. meningitidis and N. gonorrhoeae. Strains were grown overnight on GC agar supplemented with Isovitalex (BD, cat. B11876) and 10 μM deferoxamine mesylate salt (Sigma, cat. D9533) at 37 °C with 5% CO₂ and then restreaked onto several fresh GC agar supplemented with Isovitalex and 10 μM deferoxamine mesylate salt for another overnight growth. Bacteria were resuspended in PBS and heat-killed at 56 °C for a minimum of 60 min. To evaluate a potential effect of sialylation on TbpB accessibility, strains were grown overnight on GC agar supplemented with Isovitalex, sub-cultured in RPMI 1640 medium (Wisent, cat. 350-000-CL) at starting OD₅₅₀ of ~0.25 and allowed to grow at 37 °C with shaking at 160 rpm. After 1.5 h, the culture was split into two—with or without the addition of CMP-NANA to a final concentration of 50 µg/mL (Sigma, cat. 8271) and allowed to grow for an additional 1.5 h. Bacteria were pelleted (3000×g for 10 min), and pellets were resuspended in PBS for heat inactivation as above. 384-well plates were coated with 20 µL of heat-killed bacteria diluted in PBS at an OD₅₅₀ of 0.2 and dried in a biosafety cabinet until inactivation of bacteria was confirmed. Plates were blocked with 5% BSA and ELISAs were performed as described above.