ZFIN ID: ZDB-PUB-040908-19
Development of a respiratory burst assay using zebrafish kidneys and embryos
Hermann, A.C., Millard, P.J., Blake, S.L.., and Kim, C.H.
Date: 2004
Source: Journal of immunological methods   292(1-2): 119-129 (Journal)
Registered Authors: Kim, Carol H.
Keywords: none
MeSH Terms:
  • Animals
  • Embryo, Nonmammalian/immunology*
  • Embryo, Nonmammalian/metabolism
  • Fluoresceins/metabolism
  • Indoles/pharmacology
  • Kidney/immunology*
  • Kidney/metabolism
  • Maleimides/pharmacology
  • Neutral Red/metabolism
  • Phagocytes/immunology
  • Phagocytes/metabolism*
  • Protein Kinase C/antagonists & inhibitors
  • Reactive Oxygen Species/metabolism
  • Respiratory Burst*
  • Zebrafish/embryology
PubMed: 15350517 Full text @ J. Immunol. Methods
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 (H(2)DCFDA) 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 H(2)DCFDA 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-H(2)DCFDA) with the phagocyte-selective dye neutral red is consistent with the observation that macrophages and granulocytes are the ROS-producing cells in the zebrafish.