title: Development of a respiratory burst assay using zebrafish kidneys and embryos.
abstract: The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including the respiratory burst of phagocytes. Respiratory burst can be used as a reliable measure of the immune response of a host, and numerous assays have been developed to measure this response in a variety of mammal and fish species. Phagocytes, like granulocytes and macrophages, that are derived from different tissues, or grown in cell culture, have been employed in a range of assay formats employing a variety of detection methods. The small size of the zebrafish has prevented the large-scale extraction of these cells for respiratory burst assays in the zebrafish. In this work, we describe a respiratory burst assay developed for the zebrafish using intact kidneys and embryos as sources of phagocytes. Phorbol myristate acetate (PMA)-inducible reactive oxygen species (ROS) were detected following the oxidation of a non-fluorescent dye 2',7'-dihydrodichlorofluorescein diacetate (H2DCFDA) to dichlorofluorescein (DCF), a fluorescent product. Embryos from 1 day post-fertilization until 5 days post-fertilization (dpf) were employed in this assay. Abrogation of H2DCFDA oxidation by the protein kinase C (PKC) inhibitor bisindolylmaleimide I (BisI) indicated a reduction in the respiratory burst. Fluorescence from the PMA-induced respiratory burst in kidneys and embryos was significantly elevated above DMSO-treated controls, while preincubation with BisI inhibited the increase in fluorescence. Colocalization of cell-associated chloromethyl-dihydrodichlorofluorescein diacetate (CM-H2DCFDA) with the phagocyte-selective dye neutral red is consistent with the observation that macrophages and granulocytes are the ROS-producing cells in the zebrafish.
authors.affiliation: Department of Biochemistry, Microbiology, and Molecular Biology, 5735 Hitchner Hall, University of Maine, Orono, ME 04469, USA.
authors.forename: Andrea C
authors.initials: AC
authors.lastname: Hermann
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Millard
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Blake
authors.forename: Carol H
authors.initials: CH
authors.lastname: Kim
mesh_terms: D000818:Animals; D004625:Embryo, Nonmammalian / Q000276:immunology* / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D007668:Kidney / Q000276:immunology* / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D009499:Neutral Red / Q000378:metabolism; D010586:Phagocytes / Q000276:immunology / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017382:Reactive Oxygen Species / Q000378:metabolism; D016897:Respiratory Burst; D015027:Zebrafish / Q000196:embryology
chemical_list: D005452:Fluoresceins; D007211:Indoles; D008301:Maleimides; D017382:Reactive Oxygen Species; D009499:Neutral Red; C037631:2',7'-dichlorofluorescein; D011493:Protein Kinase C; C088060:bisindolylmaleimide