Generation and Characterization of Mast Cell Lines

Mice deficient in PAG and their WT littermates of C57BL/6 genotype were used in this study. PAG-KO mice were generated by the modified bacterial artificial chromosome technology as previously described (24 (link), 31 (link), 32 (link)). Bone marrow cells were isolated from the femurs and tibias of 8–12-week-old mice. The cells were cultured for 8–12 weeks in RPMI-1640 medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 71 µM 2-mercaptoethanol, minimum essential medium (MEM) non-essential amino acids, 0.7 mM sodium pyruvate, 2.5 mM l-glutamine, 12 mM d-glucose, recombinant mouse SCF (15 ng/ml, PeproTech EC), mouse recombinant IL-3 (15 ng/ml, PeproTech EC), and 10% fetal calf serum (FCS). Stable mast cell lines derived from mouse WT BMMCs (Lyn^+/+) or mouse LYN-KO BMMCs (Lyn^−/−), were obtained from Hibbs et al. (11 (link)) and were used for experiments with CSK-OE if not stated otherwise. These cell lines were cultured in RPMI-1640 medium supplemented as described above, except that SCF was omitted.