Rapid isolation of homogeneous murine bronchoalveolar lavage fluid eosinophils by differential lectin affinity interaction and negative selection.
Murine models have advanced our understanding of the immune regulation of eosinophilic inflammation but there are few methods for the reliable isolation of viable populations of eosinophils from the inflamed lung. Here we describe a method to isolate murine eosinophils in high yield and purity from lung lavage fluid after induction of eosinophilic inflammation by inhalation of ovalbumin antigen in presensitized BALB/c mice. Thirteen biotinylated plant lectins were screened for their ability to bind selectively alveolar macrophages/monocytes thus permitting the purification of eosinophils by negative selection with streptavidin-conjugated magnetic beads. Bandierea (Griffonia) simplifora isolectin I and, to a lesser extent, Jacalin, provided selective enrichment of viable eosinophils which could be further purified with biotinylated anti-lymphocyte antibodies (up to 98.5% pure). FACS analysis revealed a surface marker phenotype consistent with active effector function (Fas/CD95(+), B7-1/CD80(+), L-selectin/CD62L(Lo), ICAM-1/CD54(+), CD51(+)). Eosinophils retained functional responsiveness, responding to PMA by producing superoxide, as detected by the reduction of dihydrorhodamine-123 to rhodamine. The eosinophils were also able to undergo active apoptosis, as detected by propidium iodide DNA staining, when exposed to a cross-linking anti-Fas antibody, Jo-2. The method may be of general use in studies of murine eosinophil biology.
Shinagawa
K
K
Pharmacology Lab. Kissei Pharmaceutical Co. Ltd., 4365-1 Kashiwabara, Hotaka, Minamiazumi, Nagano, Japan. kazuhiko_shinagawa@pharm.kissei.co.jp
Anderson
G P
GP
D000280:Administration, Inhalation; D000818:Animals; D000941:Antigens / Q000008:administration & dosage; D000954:Antigens, Surface / Q000378:metabolism; D017209:Apoptosis; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology*; D004804:Eosinophils / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D005434:Flow Cytometry; D007114:Immunization; D018189:Immunomagnetic Separation / Q000379:methods*; D037102:Lectins; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010047:Ovalbumin / Q000008:administration & dosage / Q000276:immunology; D037121:Plant Lectins; D016897:Respiratory Burst; D019014:fas Receptor / Q000378:metabolism
D000941:Antigens; D000954:Antigens, Surface; C045475:Griffonia simplicifolia lectins; D037102:Lectins; D037121:Plant Lectins; D019014:fas Receptor; C043696:jacalin; D010047:Ovalbumin