Characterization of Genetically Modified Cell Lines

B16-F10 cells were originally purchased from ATCC (CRL-6475) and genetically modified to express chicken ovalbumin (OVA), eGFP, and neomycin phosphotransferase (30 (link)). The resulting OVA-expressing B16F10 cells were selected with 0.75 mg/mL G418 sulfate (ThermoFisher). HEK293 cells were a gift from Prof. Dantuma (Karolinska Institutet, Stockholm), and MC38 cells were a gift from Dr. Asis Palazon (University of Cambridge). Human umbilical vein endothelial cells (HUVEC) were obtained from ThermoFisher (C0035C). With the exception of HUVEC, cell lines were cultured in high-glucose DMEM with pyruvate (11995065, ThermoFisher) supplemented with 10% FBS (Sigma). HUVECs were cultured with EGM-2 Endothelial Cell Growth Medium (CC-3162, Lonza) containing EGM-2 SingleQuots Supplements (CC-4176, Lonza). All cells lines were cultured in the presence of 100 U/mL penicillin (Sigma) and 100 μg/mL streptomycin (Sigma) and in incubators with 5% CO₂. Except for cells obtained directly from the supplier, cell lines were initially Mycoplasma tested using the MycoAlert Mycoplasma Detection Kit (LT07–118, Lonza). Cell lines were frozen at low passage number (<5) in DMEM containing 10% DMSO (Sigma) and were typically passaged 3 to 4 times between thawing and experimental use. Cell lines were not authenticated.