Cell Line Culture Protocols

Cell lines were maintained at 37°C in the presence of 5% CO₂. Vero (American Type Culture Collection [ATCC] CCL-81), HEK293T (ATCC CRL-3216), BHK-21 (ATCC CCL-10), or ΔB4galt7 N2a (Ma et al., 2020 (link)) cells were passaged in growth medium Dulbecco's Modified Eagle Medium (DMEM [Invitrogen] supplemented with 5% [Vero and BHK-21] or 10% [HEK293T and ΔB4galt7 N2a] heat-inactivated FBS [Omega Scientific], 100 U/ml penicillin, 100 U/ml streptomycin, and 10 mM Hepes [Invitrogen]). Gibco Expi293F (Thermo Fisher Scientific) cells were maintained at 37°C in Expi293 Expression Medium. Expi293F cells were authenticated by the ATCC cell line authentication service using short tandem repeat analysis. Hybridoma cell lines were grown in Iscove's Modified Dulbecco's Medium (Thermo Fisher Scientific) supplemented with 20% FBS (Hyclone), 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 U/ml streptomycin (Invitrogen). HMMA 2.5, B95.8, and ExpiCHO cell lines were maintained as previously described (Williamson et al., 2020 (link), 2021 (link)). All cell lines were confirmed as mycoplasma negative using Washington University Induced Pluripotent Stem Cell Core Facility services or using a universal mycoplasma detection kit (ATCC 30-1012K).