Fractionation of mature eosinophils in GATA-reporter transgenic mice.
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.
Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan.
Kibom
K
Kim
Norio
N
Suzuki
Kinuko
K
Ohneda
Naoko
N
Minegishi
Masayuki
M
Yamamoto
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D000943:Antigens, Differentiation / Q000378:metabolism; D001249:Asthma / Q000097:blood / Q000276:immunology / Q000473:pathology; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D001992:Bronchoalveolar Lavage Fluid / Q000166:cytology / Q000276:immunology; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics; D002452:Cell Count; D002454:Cell Differentiation; D019070:Cell Lineage / Q000502:physiology; D002469:Cell Separation / Q000379:methods*; D003114:Colony-Forming Units Assay; D047093:Eosinophil Major Basic Protein / Q000235:genetics; D047209:Eosinophil Peroxidase / Q000235:genetics; D004804:Eosinophils / Q000166:cytology* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry / Q000379:methods; D050982:GATA1 Transcription Factor / Q000235:genetics*; D050989:GATA2 Transcription Factor / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D053666:Interleukin-5 Receptor alpha Subunit / Q000235:genetics; D007962:Leukocytes / Q000166:cytology / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008822:Mice, Transgenic; D009687:Nuclear Proteins / Q000235:genetics; D010047:Ovalbumin / Q000276:immunology; D011990:Receptors, Transferrin / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics
D015703:Antigens, CD; D000943:Antigens, Differentiation; D022763:CCAAT-Enhancer-Binding Protein-alpha; D022762:CCAAT-Enhancer-Binding Proteins; C068126:CD71 antigen; C489468:Cebpe protein, mouse; D050982:GATA1 Transcription Factor; D050989:GATA2 Transcription Factor; C494689:Gata1 protein, mouse; C494712:Gata2 protein, mouse; D053666:Interleukin-5 Receptor alpha Subunit; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D011990:Receptors, Transferrin; D014157:Transcription Factors; C107310:Zfpm1 protein, mouse; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D010047:Ovalbumin; D047209:Eosinophil Peroxidase; D047093:Eosinophil Major Basic Protein