Improved fish lymphocyte culture for chromosome preparation

Cytogenetic methodology is still underdeveloped in fishes compared with mammals. Culture condition for fish lymphocytes was optimized to improve chromosome preparation using the rainbow trout (Oncorhynchus mykiss) as a model after changing the combination of parameters such as mitogens, incubation periods, media, cell components, and freshness of blood. The optimized culture condition included isolation of lymphocytes from fresh blood by a stirring method, their culture in medium 199 supplemented with 10% FBS, 18 microg/ml of phytohemagglutinin (PHA-W) and 100 microg/ml of lipopolysaccharide (LPS) as mitogens, and harvested at 6 days after culture. This condition provided a notably increased mitotic index (MI) of 4.3-10.0% in rainbow trout lymphocytes. In addition, the condition was highly reproducible as shown by the similar level of MI in cultured lymphocytes from 181 individuals without failure. Applicability of this method in a wide range of fish groups was also proven with Ml of 1.1-13.3% in cultured lymphocytes from other 16 freshwater species of Acipenseridae, Anguillidae, Solmonidae, Cyprinidae, and Centrarchidae, and five marine species of Sparidae, Kyphosidae, Paralichthyidae, and Scorpaenidae. Chromosome preparations of improved quality by the present method were successfully applied for the replication R-banding with incorporation of 5-bromo-2'-deoxyuridine and direct R-banding fluorescence in situ hybridization.