Recombinant murine scf

BM cells were harvested by flushing femurs and tibias of β-globin/GFP transgenic mice with Roswell Park Memorial Institute (RPMI)/10% foetal bovine serum (FBS) media. Nanoparticles (2 mg ml⁻¹) were used to treat ∼300,000–500,000 cells for 48 h in RPMI/10% FBS media containing glutamine, in triplicate samples. After 48 h, cells were fixed with 4% paraformaldehyde, and analysed by flow cytometry. Cells treated with blank nanoparticles were included as a control.
For experiments with CD117+, cells were isolated by magnetic separation and grown in cells, Iscove's Modified Dulbecco's Media media containing insulin (10 ng ml⁻¹), FCS (10%) and erythropoietin (1 U ml⁻¹). Where indicated, 3 μg ml⁻¹ of SCF (recombinant murine SCF, catalogue #250-03, PeproTech, Rocky Hill, NJ;) was added before nanoparticle treatment. NPs ( 2 mg ml⁻¹) were used to treat 50,000–100,000 CD117+ cells in triplicate for 48 h in the above media, followed by flow cytometry analyses as above. Inhibitors were used at concentrations of 200 nM (dasatinib), 1.0 μM (MEK162) and 3.0 μM (BKM120). Dasatanib was obtained from Cayman Chemical (Ann Arbor, MI; item #11498) and dissolved according to manfacturer's protocol. MEK162 and BKM120 were obtained from Dr. Harriet Kluger, Yale University.

Primary mouse embryonic fibroblasts MEFs were generated and cultured according to standard protocols [45 (link)]. The Erythroid Myeloid Lymphoid (EML) cell line described previously [22 (link)] was maintained in Iscove's Modified Dulbecco's Medium (IMDM), 20% fetal bovine serum (FBS), Glutamax, penicillin-streptomycin supplemented with 100 ng/mL recombinant murine SCF (Peprotech). Human cell lines K562 [46 (link)] and M07e [27 (link)] were maintained in RPMI-1640 with glutamine and HEPES (Invitrogen), 10% FBS, penicillin-streptomycin. M07e were supplemented with 100 ng/mL recombinant human SCF and 30 ng/mL GM-CSF (Peprotech).

BMMC and BMMØ were cultured as previously described in (5 (link)). Briefly, bone marrow was isolated from femurs of wild-type and transgenic mice. To initiate differentiation of BMMCs, bone marrow cells were cultured in IMDM supplemented with 10% heat-inactivated fetal calf serum (HI-FCS), 2 mM L-Glutamine (Sigma, G7513), 100 U/ml Penicillin + 100 µg/ml Streptomycin (Sigma, N109), 50 μM β-mercaptoethanol (Sigma M6250) and 2 ng/ml murine interleukin-3 (IL-3, Peprotech 213-13). Recombinant murine SCF (5 ng/ml, Peprotech 250-03) was added only during the first passage. c-kit⁺FcεRI⁺ BMMCs were used for experiments after 4 weeks of culture. Culture medium was supplemented with IL-3 (2 ng/ml) every two days.
To generate BMMØ, bone marrow cells were cultured in bacterial petri dishes (Greiner bio-one 633180) in RPMI supplemented with 10% HI-FCS, 2 mM L-Glutamine, 1% Penicillin/Streptomycin, 50 μM β-mercaptoethanol and 20% L929-conditioned medium. Non-adherent cells were collected after 5 days of culture to perform experiments.
For BMMC and BMMØ GPCR activation adenosine (Fluka Bio Chemika 01890), recombinant mouse C5a (R&D Systems #8085-C3-025) and platelet activating factor (PAF; Sigma, P7568) were used.

We prepared mBMMCs from a 6-week-old male mouse (BALB/c) according to a previously described method [23 (link)]. Briefly, bone marrow cells were cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS) (JRH Biosciences, Lenexa, KS, USA), 10 μM 2-mercaptoethanol (Wako, Osaka, Japan), 20 mM Hepes buffer (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 100 μM MEM nonessential amino acids (Sigma-Aldrich), 2 μg/mL gentamicin solution (Sigma-Aldrich), 20 μL/mL stabilized penicillin–streptomycin solution (Sigma-Aldrich), 20 ng/mL recombinant murine interleukin-3 (IL-3; Peprotech, Rocky Hill, NJ, USA), 40 ng/mL recombinant murine SCF (Peprotech), 5 ng/mL recombinant murine IL-9 (R&D systems, Minneapolis, MN, USA), and 1 ng/mL TGF-β1 (Sigma-Aldrich) at 37 °C in a humidified 5% CO₂ atmosphere. Mast cell purity was examined by flow cytometry (FACSCalibur; Becton Dickinson, Franklin Lakes, NJ, USA), and more than 98% of the nonadherent cells were positive for FcεRI and c-kit.

Bone marrow cells were isolated from femurs and tibias of 4 to 7 week-old WT and transgenic mice. The isolated cells were cultured to obtain bone marrow-derived mast cells (BMMCs) in RPMI 1640 medium (Sigma, Cat. No. R8758) supplemented with antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin, Gibco), 71 μM 2-mercaptoethanol, minimal essential medium non-essential amino acids, 0.7 mM sodium pyruvate, 2.5 mM L-glutamine, 12 mM D-glucose, 10% fetal calf serum (FCS; Biosera, Cat. No. FB-1090/500), 15 ng/ml recombinant murine SCF (PeproTech EC, Cat. No. 250-03) and 20 ng/ml recombinant murine IL-3 (PeproTech EC, Cat. No. 213-13). Cells with Ormdl3-specific KD and corresponding controls were prepared by lentiviral transduction of 8 week-old mature BMMCs using HEK 293T/17 packaging cells for preparation of virus. Suitable short hairpin RNAs cloned into pLKO.1 vector were purchased from Open Biosystems. Equimolar mixes of TRCN0000126200 and TRCN0000126203 were used for preparation of Ormdl3 KD. Non-target pLKO.1 vector was used as a negative control^{18 (link)}.