1. Fractionation of mature eosinophils in GATA-reporter transgenic mice.
- Author
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Kim K, Suzuki N, Ohneda K, Minegishi N, and Yamamoto M
- Subjects
- Animals, Antigens, CD metabolism, Antigens, Differentiation metabolism, Asthma blood, Asthma immunology, Asthma pathology, Bone Marrow Cells cytology, Bone Marrow Cells metabolism, Bronchoalveolar Lavage Fluid cytology, Bronchoalveolar Lavage Fluid immunology, CCAAT-Enhancer-Binding Protein-alpha genetics, CCAAT-Enhancer-Binding Proteins genetics, Cell Count, Cell Lineage physiology, Colony-Forming Units Assay, Eosinophil Major Basic Protein genetics, Eosinophil Peroxidase genetics, Eosinophils immunology, Eosinophils metabolism, Eosinophils pathology, Flow Cytometry methods, Gene Expression genetics, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Interleukin-5 Receptor alpha Subunit genetics, Leukocytes cytology, Leukocytes metabolism, Luminescent Proteins genetics, Luminescent Proteins metabolism, Mice, Mice, Inbred BALB C, Mice, Transgenic, Nuclear Proteins genetics, Ovalbumin immunology, Receptors, Transferrin metabolism, Transcription Factors genetics, Cell Differentiation, Cell Separation methods, Eosinophils cytology, GATA1 Transcription Factor genetics, GATA2 Transcription Factor genetics, Genes, Reporter genetics
- Abstract
Eosinophils contribute to the pathophysiology of allergic and infectious diseases, albeit their molecular functions remain unknown. Mature eosinophils are identified by their unique morphology and staining characteristics. However, it is difficult to fractionate living eosinophils by flow cytometry because these granulocytes express multiple cell surface markers that are shared by other cells of hematopoietic or non-hematopoietic origin. In this study, we describe a flow cytometry-based method to enumerate and fractionate eosinophils by developmental stages. To fractionate these cell types, we used transgenic mouse lines that express fluorescent proteins under control of the Gata1 gene hematopoietic regulatory region (Gata1-HRD), which is exclusively active in Gata1-expressing hematopoietic cells, including eosinophils. As expected, mature eosinophils were highly enriched in the fluorescent reporter-expressing subfraction of bone marrow myeloid cells that were negatively selected by using multiple antibodies against B220, CD4, CD8, Ter119, c-Kit and CD71. Cytochemical analyses of flow-sorted cells identified the cells in this fraction as eosinophils harboring eosinophilic granules. Additionally, expression of eosinophil-specific genes, for instance eosinophil enzymes and the IL-5 receptor alpha gene, were specifically detected in this fraction. The expression of these eosinophil-specific genes increased as the cells differentiated. This method for enrichment of bone marrow eosinophils is applicable to fractionation of eosinophils and bronchoalveolar lavage fluid from transgenic mice with atopic asthma. Thus, both pathological and developmental stages of eosinophils are efficiently fractionated by this flow cytometry-based method using Gata1-HRD transgenic reporter mice. This study, therefore, proposes a useful means to study the experimental allergic and inflammatory systems.
- Published
- 2010
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