Nucleofector solution buffer

Luciferase reporter was constructed as described51 (link). Ahr promoter region (−2,000 to 0) was subcloned into pGL3 vector. Luciferase reporter vectors were cotransfected with pRL-TK (as an internal control reporter vector) into macrophages by electroporation. Luciferase assays were performed with guidelines provided by the manufacturer (Promega). For NK92 transfection, cells (1 × 10⁶) were resuspended in 100 μl Nucleofector Solution buffer (Lonza) containing 5 μg DNA, followed by transfection using the Nucleofector Program Y-001 on Amaxa nucleofector II device (Lonza). Cells were recovered in RPMI1640 media containing 4 mM L-glutamine, 1.5 g l⁻¹ sodium bicarbonate, and 10% heat-inactivated fetal bovine serum (FBS) for 6 h, followed by flow cytometry sorting for viable cells.

For BMDMs, bone marrow cells were aspirated from mouse femurs, followed by culture in RPMI 1640 media containing 10% fetal bovine serum, 50 ng ml⁻¹ macrophage colony stimulating factor (MCSF) for 7 days. For macrophage transfection, BMDMs (1 × 10⁶) were resuspended in 100 μl Nucleofector solution buffer (Lonza) containing 5 μg RNA or other substrates, followed by transfection using the Nucleofector Program Y-001 on Amaxa nucleofector II device (Lonza). Cells were recovered in RPMI 1640 media containing 4 mM L-glutamine, 1.5 g l⁻¹ sodium bicarbonate and 10% heat-inactivated fetal bovine serum for 6 h, followed by flow cytometric sorting for viable cells.

NK cell line NK92 (ATCC: CRL-2407) was cultured in Alpha Minimum Essential medium (α-MEM) containing 2 mM L-glutamine, 1.5 g l⁻¹ sodium bicarbonate, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 500 U ml⁻¹ recombinant IL-2, 12.5% horse serum and 12.5% FBS. For NK92 transfection, cells (1 × 10⁶) were resuspended in 100 μl Nucleofector Solution buffer (Lonza) containing 5 μg DNA, followed by transfection using the Nucleofector Program Y-001 on Amaxa nucleofector II device (Lonza). Cells were recovered in α-MEM for 6 h, followed by flow cytometric sorting for viable cells and further culture48 (link). For BM cell transfection, pSIN-EF2-IRES-EGFP lentiviral vectors carrying the indicated genes were transfected into HEK293T cells (maintained by our laboratory) together with psPAX2 and pMD2.G, followed by concentration through ultracentrifugation on 50,000 g for 2 h. Pellets of lentivirus were resuspended in serum-free α-MEM media. Donor BM cells were infected with lentiviruses by centrifugation at 500 g for 1.5 h in the presence of 8 μg ml⁻¹ Polybrene (Sigma-Aldrich) and then incubated at 37 °C for 18 h, followed by sorting for GFP⁺ cells that were used for transplantation into lethally irradiated recipient mice49 (link).