Cultivation and Genetic Manipulation of Mouse Mast Cells

A stable cell line of mouse bone marrow-derived mast cells (BMMCL) was generously provided by Dr. M. Hibbs (39 (link)). The cells were cultivated in freshly prepared RPMI-1640 culture medium, which was supplemented with 100 U/ml of penicillin, 100 µg/ml of streptomycin, MEM nonessential amino acids, 1 mM sodium pyruvate, 10% fetal calf serum (FCS), and 10% WEHI-3 cell supernatant as a source of interleukin-3 (IL-3). Cells were grown at 37°C in 5% CO₂ and passaged every 2-3 days. BMMCL were transfected with DNA constructs through nucleofection using a Mouse Macrophage Kit and the Amaxa Nucleofector II (program Y-001; Lonza Cologne AG, Cologne, Germany), following the manufacturer’s instructions. Subsequently, the cells were transferred to culture media supplemented with IL-3 and cultured for 24-48 hours before analysis. In some cases, cells were treated for 30 min with 100 nM Calyculin A to inhibit Ser/Thr phosphatases. Alternatively, cells were pretreated for 30 min with 10 μM LY-333531 to inhibit cPKC before Calyculin A treatment. Cells were also treated for 15 min with freshly prepared pervanadate as described previously (10 (link)) to inhibit Tyr phosphatases. To suppress the activity of Src family kinases, cells were incubated for 60 min with 20 μM PP2 before pervanadate treatment. To activate PKCs, cells were incubated for 15 min with 1 μM PMA.
The HEK 293FT packaging cells, derived from human embryonic kidney tissue, were sourced from Promega Biotec. Cells were grown at 37°C in 5% CO₂ in DMEM supplemented with 10% FCS and antibiotics. For lentivirus production, HEK 293FT cells were used at passages 4-15. Cells were transfected with DNA constructs using polyethylenimine as described previously (30 (link)).