|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.
|Source:||Journal of immunological methods 292(1-2): 119-129 (Journal)|
|Registered Authors:||Kim, Carol H.|
|PubMed:||15350517 Full text @ J. Immunol. Methods|
Hermann, A.C., Millard, P.J., Blake, S.L.., and Kim, C.H. (2004) Development of a respiratory burst assay using zebrafish kidneys and embryos. Journal of immunological methods. 292(1-2):119-129.
ABSTRACTThe 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.
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