Christine L. Densmore
Christopher A. Ottinger
Luke R. Iwanowicz
2004
<p><span>A non-specific cytotoxic <a title="Learn more about Cell Assay from ScienceDirect's AI-generated Topic Pages" href="https://www.sciencedirect.com/topics/immunology-and-microbiology/cell-assay" data-mce-href="https://www.sciencedirect.com/topics/immunology-and-microbiology/cell-assay">cell assay</a> for fish is presented that is based on the release of the activated fluorochrome <a title="Learn more about Calcein from ScienceDirect's AI-generated Topic Pages" href="https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/calcein" data-mce-href="https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/calcein">calcein</a> AM from lysed carp epithelioma <a title="Learn more about Papulosa from ScienceDirect's AI-generated Topic Pages" href="https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/papulosa" data-mce-href="https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/papulosa">papulosum</a> 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 1×10</span><sup>5</sup><span>EPC cells well</span><sup>−1</sup><span>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 <a title="Learn more about White Blood Cell from ScienceDirect's AI-generated Topic Pages" href="https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/white-blood-cell" data-mce-href="https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/white-blood-cell">leucocytes</a> 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 <a title="Learn more about Monoclonal Antibodies from ScienceDirect's AI-generated Topic Pages" href="https://www.sciencedirect.com/topics/immunology-and-microbiology/monoclonal-antibodies" data-mce-href="https://www.sciencedirect.com/topics/immunology-and-microbiology/monoclonal-antibodies">monoclonal antibody</a> 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</span><sup>−1</sup><span>of MAb5C.6 relative to no antibody (</span><i>P</i><span>≤0.013) or an equal amount of an unrelated antibody (</span><i>P</i><span>≤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 <a title="Learn more about Oncorhynchus mykiss from ScienceDirect's AI-generated Topic Pages" href="https://www.sciencedirect.com/topics/immunology-and-microbiology/oncorhynchus-mykiss" data-mce-href="https://www.sciencedirect.com/topics/immunology-and-microbiology/oncorhynchus-mykiss">rainbow trout</a> leucocytes using the </span><sup>51</sup><span>Cr-release assay.</span></p>
application/pdf
10.1016/S1050-4648(03)00056-1
en
Elsevier
Calcein AM release-based cytotoxic cell assay for fish leucocytes
article