26th Annual Eastern Fish Health Workshop

A Rhodamine-123 Based Cytotoxic Assay For Fish Leukocytes Using EPC Target Cells

Luke Iwanowicz² and Chris Ottinger¹

¹National Fish Health Research Laboratory, U. S Geological Survey, 1700 Leetown Road, Kearneysville, WV 25414; ²Johnson Controls World Services, Inc., National Fish Health Research Laboratory, U. S Geological Survey, 1700 Leetown Road, Kearneysville, WV 25414

Numerous teleost fishes including carp, catfish, rainbow trout and damselfish posses a population of natural killer-like cells referred to as nonspecific cytotoxic cells (NCC). These cells, considered analogous to mammalian natural killer (NK) cell populations, are involved with tumor cell lysis, protozoan parasite immunity and other innate immune functions. Environmental organic contaminants and some heavy metals have been associated with NCC suppression; thus, evaluating the cytotoxic activity of wild-captured fish leukocytes has been advocated as a valuable immunological biomarker. The traditional method for evaluating cytotoxicity is the ⁵¹Cr -release assay. Although very reliable, this method poses many drawbacks inherent in all radioisotopic work. Alternative non-radioisotopic methods have been developed to address this issue; however, many require the use of a flow cytometer or measure endogenous enzymes that are highly variable in viable cells. We modified a method using a fluorescent mitochondrial stain, rhodamine-123, previously documented as an adequate substitute for the ⁵¹Cr-release assay. In order to avoid the intrinsic limitations of using mammalian target cells, a fish cell line was chosen that could be cultured under atmospheric conditions and at temperatures unsuitable for mammalian cell culture. Rhodamine-123 uptake and retention properties of the epithelioma papulosum cyprini (EPC) cell line, a virally transformed line, were evaluated at various dye concentrations and incubation times at 15 and 20^oC. Target cell susceptibility to salmonid and moronid effector cells was subsequently examined and a practical cytotoxic assay developed.