A Fluorescence Based Cytotoxic Assay For Fish Leucocytes Using Calcein AM Labeled Adherent Target Cells     Luke R. Iwanowicz1, Christine L. Densmore2 and Chris A.Ottinger2   1Department of Environmental Sciences, Virginia Institute of Marines Sciences, College of William and Mary, Gloucester Point, VA; 2USGS, National Fish Health Research Laboratory, 11700 Leetown Road, Kearneysville, WV     Rainbow trout and numerous of teleost fishes posses a population of natural killer-like cells referred to as nonspecific cytotoxic cells (NCC).  These cells are involved with tumor cell lysis, protozoan parasite immunity and other innate immune functions.  The gold-standard for evaluating cytotoxicity is the 51Cr -release assay, which unfortunately possesses many of the drawbacks inherent in all radioisotopic assays.  In mammalian research, alternative non-radioisotopic methods have been developed to address this issue. We have modified a method using the acetoxymethyl ester, calcein AM, as a replacement for the conventional 51Cr label.  Calcein AM is colorless, nonfluorescent, uncharged molecule that is cleaved into a highly fluorescent product by non-specific esterases inside a target cell. We selected the carp epithelioma papulosum cyprini (EPC) cell line as a biologically relevant target. Cytotoxic activity of purified rainbow trout anterior kidney leucocytes was evaluated at three leucocyte to target cell ratios (10:1, 2:1 and 1:1) following incubation (4, 6, 8, and 10 h) with labeled EPC cells at 15oC.  Spontaneous release values were consistent with those reported with rainbow trout using the 51Cr method on the same cell line.  Peak percent cytotoxic activity value (mean ± SE n = 11 and 12 respectively) of 4.9 ± 1.11 and 11.53 ± 2.43 following 4 h and 17.2 ± 3.38 and 18.9 ± 3.47 following 8h of incubation at the 2:1 leucocyte to target cell ratio. The bimodality of the response was associated with sampling stress.