Recombinant murine stem cell factor

Bone marrow mononuclear cells isolated from the femurs and tibiae were cultured for two weeks in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and penicillin-streptomycin in the presence of 10 ng/mL recombinant murine IL-3 (PeproTech, Rocky Hill, NJ, USA) and subsequently cultured for two weeks with IL-3 and 10 ng/mL recombinant murine stem cell factor (Peprotech). The 293T cells (Thermo Fisher Scientific) were cultured in Dulbecco modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin. MEDMC-BRC6 cells [23 (link)] were purchased from RIKEN BRC (Tsukuba, Japan) and cultured in Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) containing the following: 15% FBS, ITS liquid media supplement (Sigma-Aldrich, St. Louis, MO, USA), 50 mg/mL ascorbic acid (Sigma-Aldrich), 0.45 mM α-monothioglycerol (Sigma-Aldrich), 3 ng/mL IL-3, 30 ng/mL stem cell factor (SCF), 1% penicillin-streptomycin solution and 2 mM l-glutamine (Nacalai Tesque, Kyoto, Japan).

BMMCs were obtained by culturing bone marrow cells (BMCs) from 7-week-old male BALB/c mice (CLEA Japan). BMCs, derived from femur, were cultured at 37 °C in 5% CO₂ atmosphere in Dulbecco’s modified Eagle medium (DMEM; Life Technologies) supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 µg/mL streptomycin (Thermo Fisher Scientific), 5 ng/mL interleukin-3 (R&D Systems, Minneapolis, MN, USA), 5 ng/mL recombinant murine stem cell factor (PeproTech, Rocky Hill, NJ, USA) and non-essential amino acid (Life Technologies). After 4 weeks of culture, ≥95% of cells were confirmed to be mast cells, as assessed by flow cytometric analysis of cellular staining with anti-FcεRIα conjugated FITC antibodies (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and anti-c-kit conjugated PE antibodies (Miltenyi Biotec GmbH) using FACSVerse (BD, Franklin Lakes, NJ, USA).

Both femur and tibia were surgically removed from CO₂-killed mice, and a scalpel was used to remove the epiphyses of long bones. Femurs and tibias were flushed, and bone marrow suspensions were transferred to in vitro derivation was carried out using the procedure described by Dyer et al.^{35 (link)}. Bone marrow cell cultures were incubated with recombinant murine stem cell factor (100 ng mL⁻¹, PeproTech, Rocky Hill, NJ, USA) and Flt-3 ligand (100 ng mL⁻¹, PeproTech).
On day four of culture, part of the cell culture (15 mL) was resuspended in RPMI supplemented with recombinant mouse IL-5 (rmIL-5; 10 ng mL⁻¹; R&D Systems), and incubated. Prior to incubation, cells were sampled for cytospin and DNA extraction, as well as for PCR identification of the Cre recombined allele. An additional sample was removed from cell culture for DNA extraction 2 days later. On day eight, cells were subsampled before the entire culture was resuspended in RPMI supplemented with 10 ng mL⁻¹ rmIL-5 and returned to the incubator. After cell culture media were renewed on day 10, cells were resuspended for cytospin and DNA extraction, while supernatants were collected for ELISA analysis of EPX. Prior to cell culture termination on day 12, cells were sampled for cytospin, and visual inspection of Diff-Quik-stained cytospin preparations revealed that >90–100% of total cells were eosinophils.