A lymphocyte colony assay for culture of sheep lymphocytes

Abstract

A procedure for culturing sheep peripheral blood lymphocytes in soft agar was standardized. The variables that were evaluated included the type of T lymphocyte mitogens, mitogen doses, cell number, distribution of the cells in the culture vessel and the duration of the culture. Both concanavalin A (CON A) and phytohemagglutinin A (PHA) stimulated the formation of lymphocyte colonies. However, CON A consistently produced more and larger colonies. The doses of CON A and PHA which yielded the maximum number of colonies were 35 and 61 μg/ml, respectively. The optimum number of cells per well as 1.36 × 10 6. Adherence of the cells to the floor of the culture vessel was required for the colonies to form. The maximum number of colonies was attained between day 5 and day 6 of culture. The lymphocyte colony assay may be a useful adjunct to other immunologic assays for studying cell-mediated immunity of sheep.