Cmf hbss

Chicken intestinal epithelial lymphocytes (IELs) and splenic mononuclear cells (SMCs) were isolated from jejunum and spleen, respectively, as previously described with modification [6 (link),14 (link)]. Briefly, jejunum was collected aseptically from healthy or E. necatrix-infected chicken and washed with ice-cold Ca2⁺- and Mg2⁺-free HBSS (CMF-HBSS, GE Healthcare, Pittsburgh, Pennsylvania, USA) containing 1 mM dithiothreitol (MilliporeSigma, St. Louis, Missouri, USA) followed by incubation with CMF-HBSS containing 0.5 mM EDTA and 5% FBS (GE Healthcare) for 20 min at 37°C with constant swirling. Cells released into the supernatant were pooled, passed through nylon wool (Poly Sciences, Warrington, Pennsylvania, USA), and purified by a Histopaque-1077(MilliporeSigma) density gradient method. For SMCs, collected spleen was homogenized using gentleMACS Dissociator (Miltenyi Biotec, Gaithersburg, USA) and purified by the same method described above. Freshly purified lymphocytes were cultured in complete RPMI-1640 (GE Healthcare) supplemented with 10% FBS, penicillin/streptomycin (10,000 unit/ml, Invitrogen, Carlsbad, California, USA), 50 μg/ml gentamycin, 25 mM HEPES, and 55 μM 2-Mercaptoethanol (all from MilliporeSigma). The chicken macrophage cell line HD11 was maintained in complete DMEM supplemented the same as RPMI-1640 described above.