Lentiviral Vector Production and Transduction

The Lenti-X 293T cell line (Clontech) was maintained in high-glucose Dulbecco's Modified Eagle Medium (Gibco) supplemented with 100 U/ml penicillin (Sigma-Aldrich), 100 mg/ml streptomycin (Sigma-Aldrich), 0.5% sodium pyruvate (Sigma-Aldrich), and 10% FBS (Sigma-Aldrich). Lenti X cells were cultured in 75 cm² flasks (Corning) and passed every 2–3 days. Lentiviral vectors were produced using a 3rd generation lentiviral system consisting of pMDLg/pRRE, pRSV-Rev, pMD2.G (the plasmids were a gift from Didier Trono (Addgene plasmid # 12251, 12253, 12259)), and pLV/mIL-12, plasmid encoding murine il12 gene (p35 and p40 subunits); pLV/mIL-18, plasmid encoding murine il18 gene; pLV/mIL-18/mIL-12, plasmid encoding murine il-18 and il12 gene (p35 and p40 subunits) simultaneously; and pLV/EGFP, control plasmid encoding enhanced green fluorescent protein (EGFP). Lentiviral vectors used for cell modifications were produced as previously described by Rossowska et al. [26 (link)]. LV-containing supernatant was collected and concentrated by PEG 6000 (Sigma-Aldrich) precipitation. The pellet containing lentiviral vectors was suspended in PBS and stored at -80°C. The titer of the lentiviral vectors was evaluated using the commercially available QuickTiter Lentivirus Titer Kit according to the manufacturer's instructions (Cell Biolabs).