title: Calcein AM release-based cytotoxic cell assay for fish leucocytes.
abstract: A non-specific cytotoxic cell assay for fish is presented that is based on the release of the activated fluorochrome calcein AM from lysed carp epithelioma papulosum cyprini (EPC) cells. To establish the suitability of treating EPC cells with calcein AM the uptake and spontaneous release of the calcein AM by the EPC cells was evaluated. Incubation of 5 microM calcein AM in culture medium with 1x10(5)EPC cells well(-1)for a minimum of 3 h provided sufficient labelling. Spontaneous release of fluorescence from the labelled EPC cells during 10 h of post labelling incubation ranged from 30 to 39% of the total observed fluorescence. Cytotoxic activity of trout 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 calcein AM-labelled EPC cells at 15 degrees C. In some instances, the monoclonal antibody specific for the NCC surface receptor NCCRP-1 (MAb5C.6) was included in the cultures. The activity of NCC cells was significantly inhibited in the presence of 0.25 microg well(-1)of MAb5C.6 relative to no antibody (P</=0.013) or an equal amount of an unrelated antibody (P</=0.001). Average maximum observed percent cytotoxic cell activity of approximately 18% was observed following 8 h of incubation at the 2:1 and 1:1 leucocyte to target cell ratios. Percent cytotoxic cell activity using calcein AM was similar to values reported for rainbow trout leucocytes using the 51Cr-release assay.
authors.affiliation: Virginia Institute of Marine Science, College of William and Mary, National Fish Health Research Laboratory, Kearneysville, WV 25430, USA.
authors.forename: Luke R
authors.initials: LR
authors.lastname: Iwanowicz
authors.forename: Christine L
authors.initials: CL
authors.lastname: Densmore
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Ottinger
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D003602:Cytotoxicity, Immunologic / Q000276:immunology*; D005452:Fluoresceins / Q000378:metabolism*; D005453:Fluorescence; D007694:Killer Cells, Natural / Q000276:immunology* / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000276:immunology*; D011946:Receptors, Antigen / Q000276:immunology; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D011946:Receptors, Antigen; C110644:nonspecific cytotoxic cell receptor protein 1; C085925:calcein AM