Genetically Modified B16-F10 Cell Line Protocol

B16-F10 was originally purchased from ATCC (CRL-6475) and genetically modified to express ovalbumin, eGFP, and neomycin phosphotransferase (Veliça et al., 2021 (link)). The resulting ovalbumin-expressing B16F10 cells were cultured in DMEM high glucose with pyruvate (11995065 Thermo Fisher) containing 0.75 mg/mL G418 sulphate (10131027, Thermo Fisher). HEK293 was a gift from Prof. Dantuma (Karolinska Institute, Stockholm) and cultured in DMEM high glucose with pyruvate. SKOV3 was purchased from ATCC (HTB-77) and cultured in McCoy’s 5A Medium (16600082, Thermo Fisher). Raji-GFP-Luc were purchased from Biocytogen (B-HCL-010) and cultured in complete RPMI (21875, Thermo Fisher). All media was supplemented with 1% penicillin streptomycin (15140122 Thermo Fisher) and 10% fetal bovine serum (FBS, A3160802 Thermo Fisher). Cell lines were frozen at low passage number (<5) in DMEM containing 10% DMSO and were typically passaged 3–4 times between thawing and experimental use. Cell line identities were confirmed by species-specific PCR analyses where relevant in non-human lines, as well as by functional properties. Otherwise cell lines were, as described above, obtained as authenticated lines from ATCC and were tested mycoplasma negative.