Human il 3

hMCs were generated as previously described [29 (link)] according to the ethics statement. Briefly, CD117+ haematopoietic progenitors were isolated by immunomagnetic sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) from peripheral blood mononuclear cells (PBMCs). Cells were cultured for 4 weeks in Iscove Modified Dulbecco medium (IMDM) with GlutaMAX-I supplemented with 50 µmol/L β2-mercaptoethanol, 0.5% BSA, 1% Insulin-Transferrin-Selenium, 100 U/mL penicillin, 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA), human IL-6 (50 ng/mL; PeproTech, Rocky Hill, NJ, USA), human IL-3 (10 ng/mL; PeproTech), human stem cell factor (SCF, 100 ng/mL; PeproTech), and in the above medium without IL-3 from week 4 thereafter. hMCs, cells were tested for purity and maturity after 8 weeks culture (Supplementary Figure S1). THP-1 monocyte cellular line (ATCC) was cultured in RPMI (Sigma-Aldrich, St. Louis, MO, USA) medium containing L-glutamine supplemented with 10% foetal bovine serum (FBS; Invitrogen, Waltham, MA, USA) at 37 °C and differentiated into macrophages at a concentration of 5 × 10⁵ cells/mL in a flat bottom plate using phorbol 12-myristate 13-acetate (PMA) (100 µM; Thermo Fisher Scientific, Waltham, MA, USA) for 2 h. Cells were then washed and rested in RPMI for at least 1 h before being used.

PBMNCs were harvested at a concentration of 1 × 10⁵ cells/ml and resuspended with 30% FBS/PBS 200 µl. The following recombinant human cytokines were then added to the cells: human SCF (#300-07; PeproTech) at a concentration of 66.7 ng/ml; human VEGF (#100-20; PeproTech) at a concentration of 33.3 ng/ml; human IL3 (#200-03; PeproTech) at a concentration of 13.3 ng/ml; human IGF-1 (#100-11; PeproTech) at a concentration of 33.3 ng/ml; human FGF Basic (#100-18B; PeproTech) at a concentration of 33.3 ng/ml; and, human EGF (#100-15; PeproTech) at a concentration of 33.3 ng/ml. The cell mixture was resuspended with complete MethoCult™ media (#04236; STEMCELL Technologies, Inc., Vancouver, British Columbia, Canada) at a final volume of 2 ml, and then cultured in a 37 °C environment for 14 days [11 (link)]. Endothelial progenitor cell (EPC) colony forming cells (EPC-CFCs) were assessed under phase-contrast light microscopy (Eclipse TE300; Nikon Instruments, Tokyo, Japan). A colony was defined as the presence of at least 50 cells [15 (link)]. All experiments were performed in triplicate. The numbers of colonies of PBMNCs cultured in QQ culture media and in standard culture media were compared.

hMCs were generated, as previously described.21 (link), 22 (link), 23 (link) Briefly, CD117⁺CD34⁺ cells were purified from buffy coat blood mononuclear cells by using a positive selection kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured in serum-free StemSpan medium (STEMCELL Technologies, Vancouver, British Columbia, Canada) supplemented with 100 U/mL penicillin (Invitrogen, Carlsbad, Calif), 100 μg/mL streptomycin (Invitrogen), human IL-6 (50 ng/mL; PeproTech, Rocky Hill, NJ), human IL-3 (10 ng/mL; PeproTech), human stem cell factor (100 ng/mL; PeproTech), and 10 μg/mL human low-density lipoprotein (STEMCELL Technologies). After 30 days, the cells were transferred progressively to culture medium containing Iscove modified Dulbecco medium with GlutaMAX-I, 50 μmol/L β₂-mercaptoethanol, 0.5% BSA, 1% Insulin-Transferrin-Selenium (Life Technologies, Grand Island, NY), 100 U/mL penicillin, 100 μg/mL streptomycin, human IL-6 (50 ng/mL), and human stem cell factor (100 ng/mL). After 8 to 10 weeks of culture, the cells were tested for maturity and found to be greater than 90% CD117⁺ and FcεRIa⁺ cells.
We used immunocytochemistry to characterize hMCs generated from peripheral blood precursors (details are provided in the Methods section in this article's Online Repository at www.jacionline.org).

Peripheral blood mononuclear cells (PBMCs) were obtained from buffy coats (Etablissement Français du Sang). CD34⁺ precursors cells were isolated from PBMCs (EasySep™ Human CD34 Positive Selection Kit, STEMCELL Technologies) and grown under serum-free conditions using StemSpanTM medium (STEMCELL Technologies) supplemented with recombinant human IL-6 (50 ng/ml; Peprotech), human IL-3 (10 ng/ml; Peprotech) and 3% supernatant of CHO transfectants secreting murine SCF (a gift from Dr. P. Dubreuil, Marseille, France, 3% correspond to ∼50 ng/ml SCF) for 1 week. Cells were next grown in IMDM Glutamax I, sodium pyruvate, 2-mercaptoethanol, .5% BSA, Insulin-transferrin selenium (all from Invitrogen), penicillin (100 U/ml), streptomycin (100 μg/ml) and 3% supernatant of CHO transfectants secreting murine SCF for 8 weeks then tested phenotypically (CD117+, FcεRI+) and functionally (β-hexosaminidase release in response to FcεRI crosslinking) before use for experiments. Only primary cell lines showing more than 95% CD117+/FcεRI+ cells were used for experiments.