Duck lymphocytes. II. Culture conditions for optimum transformation response to phytohaemagglutinin

The lymphocyte transformation (LT) test was performed with duck blood lymphocytes in 96-well microtitre trays against nine concentrations (0.1-100 micrograms/ml) of phytohaemagglutinin (PHA). It was found that incubation at 41.6 degrees C (duck body temperature) gave better results than incubation at 37 degrees C, and that a 6 h pulse with [3H]thymidine gave better results than an 18 h pulse. Cell concentration was very critical: successful duck LT required rather high concentrations of cells (approximately 4 X 10(6)/ml, equivalent to approximately 8 X 10(5)/culture) while lower and higher concentrations gave inferior results. Duck serum supported LT optimally when used at a concentration of 10-15%, but a serum pool from adult (approximately 6-month-old) ducks gave superior results to sera from younger birds. Foetal calf serum supported duck LT strongly when used at 20%, but only poorly at 10%, while chicken serum supported duck LT when used at 5-20%. Ultroser G was an ineffective supplement, and the addition of 5% tryptose phosphate broth to other supplements had variable effects. Using optimum culture conditions, maximum LT responses were obtained after 2-3 days' culture, when it was possible to obtain stimulation indices of 500-5000. The addition of PHA to cultures could be delayed for up to 18 h without affecting LT, but thereafter the cells rapidly lost responsiveness.