Calcein AM release-based cytotoxic cell assay for fish leucocytes

Fish and Shellfish Immunology
By: , and 



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 μM calcein AM in culture medium with 1×105EPC cells well−1for 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 °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 μg well−1of 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 ∼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.

Publication type Article
Publication Subtype Journal Article
Title Calcein AM release-based cytotoxic cell assay for fish leucocytes
Series title Fish and Shellfish Immunology
DOI 10.1016/S1050-4648(03)00056-1
Volume 16
Issue 2
Year Published 2004
Language English
Publisher Elsevier
Contributing office(s) Leetown Science Center
Description 11 p.
First page 127
Last page 137
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