TWENTY-THIRD ANNUAL EASTERN FISH HEALTH WORKSHOP JOHN CARVER INN, PLYMOUTH, MA 30 MARCH - 2 APRIL, 1998 Multiparameter Cell-Cycle Analysis Of Atlantic Salmon Peripheral Blood Leukocytes Christopher A. Ottinger2, Laura L. Brown1, Neil W. Ross1, Stewart C. Johnson1 1National Research Council of Canada, Institute for Marine Biosciences, 1411 Oxford Street, Halifax, Nova Scotia, Canada, B3H 3Z1; 2National Fish Health Laboratory, 1700 Leetown Road, Kearneysville, West Virginia, USA There is an increasing need for techniques that yield highly detailed information on leukocyte function. FACS-based cell cycle analysis was applied to mitogen-stimulated Atlantic salmon peripheral blood leukocytes (PBLs). Cell cycle analysis using only the nucleic acid stain propidium iodide revealed an increased percentage of cells at the G2/M stage in mitogen-stimulated cultures. However, factors such as high background staining of mitogen-induced mRNA made this approach undesirable. To address these problems a multiparameter method which used both the presence of proliferating cell nuclear antigen (PCNA/cyclin) and the amount of DNA in the cells as quantified by propidium iodide was employed to identify proliferating cells. PCNA/cyclin is a highly conserved protein and is know to be expressed in salmonids. Using Western blot analysis of reducing SDS-PAGE gels we evaluated the ability of a commercially available anti-PCNA/cyclin mAb developed against mammalian PCNA/cyclin to recognize this protein in the PBLs. The molecular weight of the protein recognized by the mAb was consistent with established value for PCNA/cyclin in mammals. The occurrence of PCNA/cyclin within salmon PBLs was confirmed by epifluorescence microscopy comparing mAb-associated FITC activity in the nucleus of PBLs from mitogen-stimulated and non-stimulated cultures. The multiparameter method consists of paraformaldehyde fixation followed by Zwittergent permeablization of the cells. Prior to the addition of the propidium iodide, the PBLs were treated with RNAase to reduce background staining. This multiparameter method allows for both traditional proliferation measures (mitogenic indices based on the percentage of PBLs expressing PCNA/cyclin in mitogen treated and non-treated cultures), detailed analysis of cell cycle position, and evaluation of apoptosis.   Return to 23rd Annual Eastern Fish Health Workshop Return to Leetown Science Center Home Page