title: Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations.
abstract: Flow cytometry is a common approach to study invertebrate immune cells including earthworm coelomocytes. However, the link between light-scatter- and microscopy-based phenotyping remains obscured. Here we show, by means of light scatter-based cell sorting, both subpopulations (amoebocytes and eleocytes) can be physically isolated with good sort efficiency and purity confirmed by downstream morphological and cytochemical applications. Immunocytochemical analysis using anti-EFCC monoclonal antibodies combined with phalloidin staining has revealed antigenically distinct, sorted subsets. Screening of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between amoebocytes and eleocytes, with the former being in favor of bacterial engulfment. This study has proved successful in linking flow cytometry and microscopy analysis and provides further experimental evidence of phenotypic and functional heterogeneity in earthworm coelomocyte subsets.
authors.affiliation: Department of Immunology and Biotechnology, Clinical Center, University of Pécs, Pécs, Hungary. Electronic address: engelmann.peter@pte.hu.
authors.forename: Péter
authors.initials: P
authors.lastname: Engelmann
authors.affiliation: Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Hayashi
authors.affiliation: Department of Immunology and Biotechnology, Clinical Center, University of Pécs, Pécs, Hungary.
authors.forename: Kornélia
authors.initials: K
authors.lastname: Bodó
authors.affiliation: Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, University of Pécs, Pécs, Hungary; János Szentágothai Research Centre, Pécs, Hungary.
authors.forename: Dávid
authors.initials: D
authors.lastname: Ernszt
authors.affiliation: Department of Comparative Anatomy and Developmental Biology, Faculty of Sciences, University of Pécs, Pécs, Hungary.
authors.forename: Ildikó
authors.initials: I
authors.lastname: Somogyi
authors.affiliation: Department of Comparative Anatomy and Developmental Biology, Faculty of Sciences, University of Pécs, Pécs, Hungary.
authors.forename: Anita
authors.initials: A
authors.lastname: Steib
authors.affiliation: János Szentágothai Research Centre, Pécs, Hungary; Department of Biophysics, Faculty of Medicine, University of Pécs, Pécs, Hungary.
authors.forename: József
authors.initials: J
authors.lastname: Orbán
authors.affiliation: Department of Comparative Anatomy and Developmental Biology, Faculty of Sciences, University of Pécs, Pécs, Hungary.
authors.forename: Edit
authors.initials: E
authors.lastname: Pollák
authors.affiliation: János Szentágothai Research Centre, Pécs, Hungary; Department of Biophysics, Faculty of Medicine, University of Pécs, Pécs, Hungary; MTA-PTE Nuclear-Mitochondrial Interactions Research Group, Pécs, Hungary.
authors.forename: Miklós
authors.initials: M
authors.lastname: Nyitrai
authors.affiliation: Department of Immunology and Biotechnology, Clinical Center, University of Pécs, Pécs, Hungary.
authors.forename: Péter
authors.initials: P
authors.lastname: Németh
authors.affiliation: Department of Comparative Anatomy and Developmental Biology, Faculty of Sciences, University of Pécs, Pécs, Hungary.
authors.forename: László
authors.initials: L
authors.lastname: Molnár
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D002469:Cell Separation; D000067493:Dynamic Light Scattering; D005434:Flow Cytometry / Q000379:methods*; D005944:Glucosamine / Q000378:metabolism; D007150:Immunohistochemistry; D016130:Immunophenotyping; D037102:Lectins / Q000378:metabolism; D008853:Microscopy / Q000379:methods*; D009835:Oligochaeta / Q000276:immunology*; D010586:Phagocytes / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D037102:Lectins; D005944:Glucosamine
keywords: Cytochemistry; FACS; Lectin binding; Monoclonal antibody; Phagocytosis