A multicolour flow cytometry identifying defined leukocyte subsets of rainbow trout (Oncorhynchus mykiss).

The investigation of the cellular immune response in fish species has been for a long time hampered by absence of appropriate monoclonal antibodies (MAbs) recognising subset specific surface markers. Consequently, the majority of immunological studies still focus on the changes in total leukocyte numbers or describe gene pattern in lymphoid organs without any information about their cellular composition. Flow cytometric techniques are routinely used for the evaluation of the leukocyte composition in numerous vertebrate species and contributed significantly to the current knowledge of immune system. In rainbow trout is so far only a limited number of MAbs against characterised (IgM and IgT, CD8α) or unknown lineage markers on thrombocytes, myeloid cells or T cells available. By combination of several MAbs, we developed a rapid, simple, accurate and high throughput method for reliable discrimination of major leukocyte subpopulations from 10 μl of peripheral blood. Additionally, by a consecutive gating, this mixture enables the evaluation of the proportion between CD8α(+) and CD8α(-) population and provides for the first time valuable information about the kinetic of CD4(+) cells in rainbow trout. Furthermore, the combination of all antibodies within one sample reduced the hands-on time down to 90 min allowing fast and accurate estimation of cell kinetics in a high number of individuals. Thus presented findings enable the precise evaluation of the cellular components of immune system during both pathological and physiological responses and have therefore an immense potential for future applications in the development of vaccines and better understanding of fish immune system.