Measurement of in vitro leucocyte mitogenesis in fish: ELISA based detection of the thymidine analogue 5'-bromo-2'-deoxyuridine.
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 (3)H-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.
Gauthier
David T
DT
Department of Environmental Sciences, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, VA 23062, USA.
Cartwright
Deborah D
DD
Densmore
Christine L
CL
Blazer
Vicki S
VS
Ottinger
Christopher A
CA
D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism; D002455:Cell Division / Q000276:immunology; D003208:Concanavalin A / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D007202:Indicators and Reagents / Q000378:metabolism; D007962:Leukocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D017686:Oncorhynchus mykiss / Q000276:immunology* / Q000378:metabolism; D010471:Perches / Q000276:immunology* / Q000378:metabolism; D011043:Pokeweed Mitogens / Q000494:pharmacology
D007202:Indicators and Reagents; D011043:Pokeweed Mitogens; D003208:Concanavalin A; D001973:Bromodeoxyuridine