Mouse recombinant interleukin il 3

All animals were sacrificed in accordance
with the Canadian Council on Animal Care Guidelines and Policies (https://ccac.ca/en/about-the-ccac/) with approval from the Health Science Animal Care and Use Committee
for the University of Alberta. Femurs were removed from 12 week-old
C57Bl/6 mice (a kind gift from Dr. Troy Baldwin, University of Alberta)
using standard dissection. Bone marrow was aspirated using a 27 gauge
needle, and the cells were cultured in RPMI media (Fisher, Hampton,
NH, USA) supplemented with 4 mM l-glutamine (Fisher), 50
μM β-mercaptoethanol (BME, Sigma-Aldrich, Oakville, ON,
Canada), 1 mM sodium pyruvate (Fisher), 100 U/mL penicillin/100 μg/mL
streptomycin (Fisher), 0.1 mM nonessential amino acids (Fisher), 25
mM HEPES (Fisher), 10% FBS (Gibco, Burlington, ON, Canada), and 30
ng/mL mouse recombinant interleukin (IL)-3 (PeproTech, Rocky Hill,
New Jersey, USA), pH-7.4–7.6, in a humidified atmosphere of
5% CO₂ in air at 37 °C. This media will be referred
to as “supplemented RPMI”. The cell suspensions were
maintained at a density of 0.5 × 10⁶ cells/mL for
4 weeks when the cells were tested for FcεRI and c-Kit expression
by flow cytometry to confirm maturation.

Mice were euthanized by CO₂ asphyxiation following isoflurane anesthesia. The tibia and femur were isolated and whole bone marrow was harvested. All animal studies were conducted in accordance with the Canadian Council on Animal Care Guidelines and Policies (https://ccac.ca/en/about-the-ccac/) with approval from the Health Science Animal Care and Use Committee for the University of Alberta. Bone marrow was aspirated using a 19 gauge needle and the cells were cultured in RPMI media (Fisher, Hampton, New Hampshire, USA) supplemented with 4 mM L-glutamine (Fischer), 50 μM BME (Sigma-Aldrich, Oakville, Ontario, Canada), 1 mM sodium pyruvate (Fischer), 100 U/ml penicillin/100 μg/ml streptomycin (Fischer), 0.1 mM nonessential amino acids (Fischer), 25 mM HEPES (Fisher), 10% FBS (Gibco, Burlington, Ontario, Canada) and 30 ng/ml mouse recombinant interleukin (IL)-3 (Peprotech, Rocky Hill, New Jersey, USA), pH-7.4–7.6, in a humidified atmosphere of 5% CO₂ in air at 37 °C. This media will be referred to as “supplemented RPMI”. The cell suspensions were maintained at a density of 10⁵ cells/ml for 4 weeks when the cells were tested for FcεRI and c-Kit expression by flow cytometry to confirm maturation. After 4 weeks, 99% of cells were double positive for c-Kit and FcεRI. BMMC were used between 4 and 8 weeks of age.