Recombinant mouse il 3

Bone marrow-derived mast cells were cultured from femoral marrow cells of C57Bl/6 mice (Jackson Laboratories, Bar Harbor, ME). Bone marrow from 2 mice were used for each batch of mast cells. Cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Primocin^™ (Invivogen, San Diego, CA), 25 mM HEPES, 1.0 mM sodium pyruvate, nonessential amino acids (BioSource International, Camarillo, CA), 0.0035% 2-ME and 300 ng/ml recombinant mouse IL-3 (PeproTech, Rocky Hill, NJ). Mast cells were used following 4–6 weeks of culture at 37°C and 5% CO₂. All animal procedures were conducted in accordance with the National Institutes of Health guidelines and approved by the University of Colorado Denver Institutional Animal Care and Use Committee. All animals were treated humanely and with regard for alleviation of suffering. Cytotoxicity of AgNPs at concentrations used in this study were evaluated by MTS and LDH assays (Promega, Madison WI) and did not induce cytotoxicity compared to the control group (data not shown). Further these concentrations were based on the evaluation of other nanoparticles which have utilized similar concentrations as performed by the NIEHS Nano GO Consortium (Xia et al., 2013 (link)).

The preparation of bone marrow derived mast cells was done as previously described (Kalesnikoff and Galli, 2011 (link)). The purity of MCs was >90% based on toluidine blue staining and surface expression of CD117 and FcεRI. For adoptive transfer of mast cells in Kit^W-sh/W-sh mice, wild type (WT) mast cells were obtained after a 4–5 week culture of bone marrow cells in medium containing 20 ng/ml recombinant mouse IL-3 (Peprotech). For MC-reconstitution studies, bone marrow derived mast cells were adoptively transferred via subcutaneous injection of 10⁶ cells into eight sites in the shaved dorsal skin of 6–8 wk Kit^W-sh/W-sh mice. After 6 weeks, skin was collected for analysis of EDC gene expression.

Bone marrow cells from constitutive or inducible knockout mice (for a list of genetic mouse models used in this study see Supplementary Table 4) were harvested and cultured in Iscove’s modified Dulbecco’s medium (IMDM, Invitrogen, Carlsbad, CA) with GlutaMAX containing 20% fetal bovine serum, 50 μM 2-mercaptoethanol, 100 IU/ml penicillin, 100 μg/ml streptomycin in the presence of cytokines. For pre-B cell culture, bone marrow cells were cultured in IMDM with 10 ng/ml recombinant mouse IL-7 (Peprotech, Rocky Hill, NJ) on OP9 stroma cells. For ALL leukemia model, pre-B cells were retrovirally transduced by BCR-ABL1. ALL cells generated from inducible knockout mice were retrovirally transduced with ERT2 or Cre ERT2 virus, and puromycin selection was performed. 4-OHT was used to induce Cre mediated gene deletion. For CML-like leukemia model, the myeloid-restricted protocol described previously was used^{23 (link)}, which generates CML-like cells. Briefly, bone marrow cells were cultured in IMDM with recombinant mouse IL-3 (10 ng/ml), IL-6 (25 ng/ml), SCF (50 ng/ml, PeproTech, Rocky Hill, NJ) and then transformed by BCR-ABL1 retroviurs. Cytokines were removed after BCR-ABL1 transduction.