Umnsah df 1

Human epithelial carcinoma cells (HeLa CCL2; ATCC), for viral replication, transfection, and rescue methods, were grown and maintained in Dulbecco Modified Eagle Medium (DMEM; Thermo Fisher, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA, USA), 100 μg of penicillin/mL, and 0.1 μg of streptomycin/mL (Invitrogen, Carlsbad, CA, USA), in a 5% CO₂ incubator at 37 °C. The DF-1 chicken embryo fibroblast cell line, which was utilized for viral rescue and virus neutralization assays, (UMNSAH/DF-1; ATCC) was maintained in DMEM supplemented with 10% FBS. For use in rLAS-uPA in vitro cytotoxicity studies, canine J3T and human U-87 (U-87 MG; ATCC) glioma cell lines were cultured in RPMI-1640 (Cellgro, Mediatech, Manassas, VA, USA) with supplemental 10% FBS in a 5% CO₂ incubator at 37 °C. The canine J3T cell line was obtained from Dr. Michael Berens (Barrow Neurological Institute, Phoenix, AZ, USA) [15 (link)].

Chicken DF-1 fibroblast cells (UMNSAH/DF-1, CRL-12203, ATCC) were maintained in high glucose DMEM supplemented with 10% fetal bovine serum (Hyclone) and 1× ABAM. Cells were maintained at 37 °C at 5% CO₂ under 60–70% relative humidity. To establish cell lines that stably overexpress the genes of interest, 1.2 μg of overexpression vector and 0.8 μg of piggyBac transposon (pCyL50) were transfected into DF-1 cells using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). After 6 h, transfection mixtures were replaced with DF-1 culture medium supplemented with puromycin (Thermo Fisher Scientific). Cells were maintained in culture medium supplemented with puromycin for 1 week to recover overexpressing cells. The cell lines were maintained for more than 15 passages for subsequent analysis.

DF-1 cells, a spontaneously transformed chicken fibroblast cell line (UMNSAH/DF-1 (ATCC # CRL-12203)) and BHK-21 cells (ATCC # CCL-10) were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia) and were grown in Dulbecco’s Minimal Essential Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 0.05 mg/mL gentamicin. MVA, a gift from Dr. Linda Wyatt and Dr. Bernard Moss (NIAID/NIH), and recombinant MVA vectors were prepared and titered in DF-1 cells.