Mycoalert plus detection kit

HEK293T, PaTu-8988T, PANC-1, C2C12, and 3T3-L1 cells were purchased from the Cell Resource Center and authenticated by authentication testing (Chinese Academy of Sciences, Shanghai, China). All cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich) containing 12% calf serum (Sigma-Aldrich), 100 U/mL penicillin (Beyotime Biotechnology, Shanghai, China), 100 μg/mL streptomycin (Beyotime Biotechnology), and 0.25 μg/mL amphotericin B (Sangon Biotech, Shanghai, China) at 37 °C in a 5% CO₂ incubator (Thermo Fisher Scientific, Waltham, MA, USA). During metabolic studies, all cells were tested for mycoplasma and declared mycoplasma-free using a MycoAlert™ PLUS detection kit (Lonza Corporation, Basel, Switzerland).

The murine cell lines CT26.WT (ATCC CRL-2638) (colon carcinoma, female), B16-F10 (CRL-6475) (malignant melanoma, male) and the human cell line SJSA-1 (CRL-2098) (osteosarcoma, male) were purchased from ATCC. At arrival, the cell lines were expanded and aliquots were frozen at low passage numbers. None of the cell lines were further authenticated. B16-F10 p53^−/−(clone 8) was generated from the B16-F10 cell line,⁴⁰ (link) expanded and frozen.
Prior to experiments, the cell lines were thawed and allowed to adjust to culture for a minimum of one week, until normal growth rate was obtained. All cell lines were grown in RPMI-1640 medium (R8758 Sigma Aldrich) supplemented with 10% heat inactivated FBS (SV30160.03, Hyclone) and 100 U/mL penicillin and 100 μg/mL streptomycin (5140-122, Gibco). The cell lines were sub-cultivated before reaching confluency, at least twice per week. All cell cultures were maintained at 37°C and 5% CO₂. None of the cell lines were maintained in culture for more than five months.
The cultures were tested and confirmed to be negative for mycoplasma infection every second month, using the MycoAlert Plus Detection kit (LT07-710, Lonza) according to the manufacturer’s instructions.

To establish a murine melanoma lung model, B16F10 cells (ATCC, Manassas, VA, USA, Cat#CRL-6475) were confirmed to be mycoplasma-free (MycoAlert PLUS detection kit, Lonza, Basel, Switzerland, Cat#LT07-705) and grown in DMEM supplemented with a 10% HI bovine calf serum (VWR International, Mississauga, ON, Canada, Cat#2100-500) in a humidified incubator at 5% CO₂ and 37 °C. Then, 3 × 10⁵ cells were injected into the tail vein of the mice. The mice were then injected intravenously with phosphate-buffered saline (PBS; control group) or 5 × 10⁷ plaque-forming units (PFUs) of OrfV in 100 μL of PBS (treatment group), four days following the tumor challenge. For the survival experiments, the mice were monitored until the signs of respiratory distress and illness were observed, following which they were euthanized. For the visualization of lung tumor burdens, the mice were euthanized 14 days post-tumor challenge, and their lungs were harvested. The number of superficial lung metastases was determined by the separation of lung lobes and enumeration under a dissection microscope (Leica, Richmond Hill, Ontario, Canada).