Recombinant human ccl11

Granulocytes were isolated from peripheral blood of allergic or healthy donors. Eosinophils were enriched and purified by negative selection using the human eosinophil enrichment cocktail (SSep™, StemCell Technologies, Seattle, WA, USA) and the MACS bead procedure (Miltenyi Biotec, Auburn, CA, USA), as previously described (23 (link)) with the exception that hypotonic red blood cell (RBC) lysis was omitted to avoid any potential for RBC lysis to affect eosinophil function. Eosinophil viability and purity were greater than 99% as determined by ethidium bromide (Molecular Probes, Life Technologies, Carlsbad, CA, USA) incorporation and cytocentrifuged smears stained with HEMA 3 stain kit (Fisher Scientific, Medford, MA, USA), respectively. Purified eosinophils (10⁶ cells/mL) were stimulated with TNF-α (200 ng/mL; R&D Systems, Minneapolis, MN, USA) or recombinant human CCL11 (100 ng/mL; R&D Systems), in RPMI-1640 medium plus 0.1% ovalbumin (Sigma, St. Louis, MO, USA), or medium alone at 37°C, for 1 h. At these concentrations, CCL11 and TNF-α induce consistent cell secretion (16 (link)).

Granulocytes were isolated from peripheral blood of allergic or healthy donors. Eosinophils were enriched and purified by negative selection as previously described (StemSep^TM, StemCell Technologies, Seattle WA; Miltenyi Biotec, Auburn, CA; Bandeira-Melo et al., 2000 (link); Akuthota et al., 2014 (link)). The hypotonic red blood cell (RBC) lysis was omitted to avoid any potential for RBC lysis to affect eosinophil function. Eosinophil viability and purity were >99% as determined by ethidium bromide (Molecular Probes, Life Technologies, Carlsbad, CA) incorporation and cytocentrifuged smears stained with HEMA 3 stain kit (Fisher Scientific, Medford, MA), respectively. Purified eosinophils (10⁶ cells/mL) were stimulated with TNF-α (200 ng/mL; R&D Systems, Minneapolis, MN) or recombinant human CCL11 (100 ng/mL; R&D Systems), in RPMI-1640 medium plus 0.1% ovalbumin (OVA; Sigma, St. Louis, MO, USA), or medium alone at 37°C, for 1 h as before (Carmo et al., 2016 (link)).