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Isolation of leucocytes from the anterior kidney and spleen of rainbow trout in a self‐generating density gradient
Author(s) -
Congleton J. L.,
Greenlee A. R.,
Ristow S. S.
Publication year - 1990
Publication title -
journal of fish biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.672
H-Index - 115
eISSN - 1095-8649
pISSN - 0022-1112
DOI - 10.1111/j.1095-8649.1990.tb03558.x
Subject(s) - spleen , biology , rainbow trout , centrifugation , phagocytosis , differential centrifugation , density gradient , microbiology and biotechnology , immunology , fish <actinopterygii> , biochemistry , fishery , physics , quantum mechanics
Separation of leucocytes from tissues and enrichment of specific types of leucocytes are essential first steps in studies of leucocyte function. We describe a simple and rapid method for separating and enriching leucocytes from the anterior kidney and spleen of rainbow trout. Leucocytes were separated on self‐generating density gradients of Sepracell‐MN, a colloidal silica‐based medium. Recovery of leucocytes from the gradients was near 100% and was not affected by procedural variables such as cell suspension: Sepracell‐MN ratio (separation ratio), initial temperature, centrifugation time, or number of cells per gradient. Two bands of leucocytes formed at separation ratios of 1 : 1, 1:1.5, 1:2 and 1 : 3. Recovery of selected leucocyte types could be maximized, and contamination by other cell types reduced, by selection of the appropriate separation ratio and cell band. Recovered leucocytes were responsive in assays of functional capability (adherence, phagocytosis, superoxide anion production). Leucocyte populations that adhered to glass were enriched for macrophages, but some neutrophils and lymphocytes (paricularly spleen lymphocytes) were also adherent. The most abundant leucocytes in the anterior kidney and spleen of the experimental fish were lymphocytes (respectively, 47 and 88%, including thrombocytes), neutrophils (35 and 7%), and macrophages (11 and 3%).