Mycoplasma detection kit

The mouse bladder cancer MB49 cell line was used to investigate the in vitro and in vivo functions of IFN-λ3.27 (link) Cells were tested yearly for mycoplasma contamination using a mycoplasma detection kit (Yeasen, China). The mouse Ifnl3 plasmid was purchased from IGEbio (China). For stable transfection, HEK-293T-derived lentivirus was used in conjunction with polybrene (Beyotime, China). Specifically, HEK-293T packaging cells were seeded at a density of 1×10⁶ cells per medium-sized dish. The HEK-293T cells were transfected with 3 µg pCDH- Ifnl3 vector, 2.25 µg psPAX2, and 0.75 µg pMD2G using 400 µl Opti-MEM and 9 µl x-tremeGENE (Roche, Switzerland). After collecting the supernatant with the viral particles over 48 hours, it was filtered through a 0.45 µm syringe filter and concentrated using 4 M NaCl and PEG 8000. The viral particles were resuspended in phosphate buffered saline (PBS) and stored at –80°C following centrifugation at 3200 g for 20 min. The cells were cultured at 37℃ under 5% CO₂ in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, ExCell Bio, China), 100 units/mL penicillin, and 100 µg/mL streptomycin (Gibco, USA). In some experiments, MB49 cell line was stimulated with 100 ng/mL recombinant mouse IFN-λ3 (rmIFN-λ3, Novus biologicals, USA) for 16 hours to evaluate its inflammatory response.