Ficoll hypaque

Blood samples were taken by venipuncture into sterile, EDTA tubes. PBMCs were isolated by density gradient centrifugation on Ficoll-Hypaque (Millipore Sigma) at 2000 rpm at room temperature for 30 min. Interphase cells were removed and washed twice in PBS. The cells were counted with a hemocytometer using Türk’s solution (Millipore Sigma) and resuspended in FACS buffer (0.1% sodium azide, 1% human AB serum, 1% FBS in PBS).

Mouse bone marrow cells were harvested from WT or β2-AR^−/− mice by flushing femurs with sterile PBS. Human PBMCs were isolated from healthy volunteer donors by venipuncture and subsequent differential density gradient separation (Ficoll Hypaque, MilliporeSigma) was carried out. Bone marrow cells were then cultured in RPMI supplemented 10% FBS, 1% l-glutamine, 1% penicillin/streptomycin, IL-6 (20 ng/mL, Biolegend), and GM-CSF (20 ng/mL, Biolegend) for 7 days, in the presence or absence of ISO (10 μM). Media, cytokine and ISO were refreshed twice during generation, and adherent cells were removed using the Detachin Cell Detachment Solution (Genlantis, catalog T100100). Bone marrow derived murine MDSCs were characterized using CD11b, Ly6G, Ly6C and Gr-1 markers. Gr-1 positive cells were used for in vitro experiments. Human MDSC populations were characterized by flow cytometry using the markers CD14 and CD33, and CD33⁺ cells were isolated from each culture using EasySep HLA Chimerism CD33 Whole Blood Positive Selection Kit (STEMCELL Technologies) per manufacturer’s instructions. The purity of isolated cell populations was determined to be greater than 90% by flow cytometry.

Peripheral blood mononuclear cells (PBMCs) were isolated through Ficoll-Hypaque (Sigma) density centrifugation from blood obtained from healthy volunteers. CD34⁺ cells were enriched from PBMCs using positive magnetic affinity column purification (Miltenyi Biotec) and eosinophil progenitors were derived using the technique of Hudson et al. (22 (link)) by culturing purified CD34⁺ cells in complete medium (RPMI1640 and 10% FBS) supplemented with stem cell factor (SCF; 25 ng/ml; BD Biosciences), thymopoietin (TPO; 25 ng/ml; R&D System, Minneapolis, MN), Fms-like tyrosine kinase 3 (Flt3) ligand (25 ng/ml; BD Biosciences), IL-3 (25 ng/ml; BD Biosciences) and IL-5 (25 ng/ml; BD Biosciences) with or without IFN-γ (20 ng/ml; BD Bioscience) for 3 days and then cultured for an additional 3 weeks with just IL-3 and IL-5 (±IFN-γ). Cells were washed and fresh media and cytokines were applied weekly. Maturation was accessed as previously described (17 (link)) and cells were activated as described above.
Mast Cells. For studies involving mast cells, CD34⁺ cells isolated as described were cultured for 8 weeks with stem cell factor and IL-6 and, for the first week only, with IL-3 according to the methodology of Kirshenbaum et al (23 (link), 24 (link)).