Measurement of in vitro leucocyte mitogenesis in fish: ELISA based detection of the thymidine analogue 5′-bromo-2′-deoxyuridine

David T. Gauthier, Deborah D. Cartwright, Christine L. Densmore, Vicki S. Blazer, Christopher A. Ottinger

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

In this study we present a method for the measurement of in vitro mitogenesis in fish leucocytes that is based on the incorporation of the thymidine analogue 5′-bromo-2′-deoxyuridine (BrdU) into the DNA of replicating cells, followed by ELISA-based detection. This technique, adapted from methods developed for mammalian cells, operates on a similar biological principle to 3H-thymidine incorporation, but circumvents the logistical and safety issues inherent with the radioactive label. Because it directly measures DNA proliferation, the assay has advantages over other colorimetric methods that may be strongly influenced by leucocyte metabolic status. Using BrdU incorporation followed by ELISA, we evaluate the responsiveness of rainbow trout (Oncorhynchus mykiss [Walbaum]) leucocytes to the mammalian T-cell mitogen Concanavalin A (Con A) as well as the differential response of white perch (Morone americana [Gmelin]) leucocytes to Con A and pokeweed mitogen. Specific considerations intrinsic to the assay system are discussed, including the implications of utilising enzyme-based detection.

Original languageEnglish (US)
Pages (from-to)279-288
Number of pages10
JournalFish and Shellfish Immunology
Volume14
Issue number4
DOIs
StatePublished - Apr 2003

All Science Journal Classification (ASJC) codes

  • Environmental Chemistry
  • Aquatic Science

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