Recombinant mouse stem cell factor scf

Generation of 4.1R-KO mice and their backcrossing onto the C57BL/6 background has been described (38 (link)). Mice were bred and maintained at the Institute of Molecular Genetics in a specific pathogen-free facility and used in compliance with the Institute guidelines.
BMMCs were derived from stem cells in the femurs and tibias of 6–8-week-old 4.1R-KO mice or their WT littermates. The cells were cultured for 8–12 weeks in RPMI-1640 culture medium supplemented with 10% fetal calf serum, minimum essential medium non-essential amino acids, 0.7 mM sodium pyruvate, 2.5 mM L-glutamine, 12 mM D-glucose, antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin), 71 μM 2-mercaptoethanol, recombinant mouse stem cell factor (SCF; 15 ng/ml, PeproTech EC), and recombinant mouse IL-3 (15 ng/ml, PeproTech EC).

BMMCs were cultured from the bone marrow cells (BMCs) of rats as previously described [19 (link)]. Briefly, BMCs were cultured for up to 10 weeks in enriched RPMI-1640 medium (containing 100 U/ml penicillin, 100 μg/ml streptomycin, 25 mmol/l HEPES, 2 mmol/l L-glutamine, 1 mmol/l sodium pyruvate, 0.1 mmol/l nonessential amino acids, 0.05 mmol/l β-ME, and 10% FBS) in the presence of both recombinant rat IL-3 (5 ng/ml, R&D Systems Inc.) and recombinant mouse stem cell factor (SCF, 5 ng/ml, PeproTech). The nonadherent cells were hemidepleted twice each week with enriched medium containing the cytokines mentioned above. After 3 weeks, >98% of the cells in the culture were MCs as determined by staining with toluidine blue.

Reagents used in this study were acquired from the indicated suppliers: trans-resveratrol, cyclodextrin, 5Z-7-Oxozeaenol and Evans blue (Sigma-Aldrich, St. Louis, MO, USA); LY294002, wortmannin, and ICI 182,780 (Abcam, Cambridge, UK); PF-3644022 (TOCRIS Bioscience, Bristol, U.K.); recombinant mouse IL-3, recombinant mouse stem cell factor (SCF), and recombinant human IL-33 (PeproTech, Rocky Hill, NJ, USA); recombinant human IL-3 (Thermo Fisher Scientific, Wilmington, DE, USA); recombinant mouse IL-33 (R & D systems, Minneapolis, MN, USA); anti-TNP IgE, anti–DNP mouse IgE mAb, anti–mouse CD16/32, PE–conjugated anti–mouse c-kit Ab, and APC–conjugated anti–mouse ST2 Ab (BD Bioscience, San Jose, CA, USA); DNP–BSA (Cosmo Bio, Tokyo, Japan); APC–conjugated anti–mouse CD63 Ab (Miltenyi Biotec, Bergisch Gladbach, Germany); anti–phospho–transforming growth factor β-activated kinase 1 (TAK1) Ab (Thr184/187; #4508), anti–phospho–IκB kinase (IKK) α/β Ab (Ser176/177; #2078), anti–phospho–p65 Ab (Ser536; #3033), anti–phospho–p38 Ab (Thr180/Thy182; #4511), anti–phospho–MK2 Ab (Thr334; #3007), anti–phospho–Akt Ab (Ser473; #4060), anti–phospho–Gab2 Ab (Tyr452; #3882), anti–phospho–Syk Ab (Tyr525/526; #2710), anti–phospho–p70S6K Ab (Tyr389; #9234), anti–phospho–AMPK Ab (Thr172; #2535), anti–β-actin Ab (#4970), and AICAR (#9944) (Cell Signaling Technology, Danvers, MA, USA).