Development of optimal conditions for the stimulation of chicken peripheral blood lymphocytes by phytohaemagglutinin (PHA) in the microculture system.

A variety of parameters influencing phytohaemagglutinin (PHA) stimulation assays of lymphocytes obtained from chicken peripheral blood have been studied. The parameters examined were PHA preparation and dilution, source and quantity of serum, cell concentration, culture medium, incubation temperature, length of the incubation period, cell separation procedure and individual influence of the blood donors. Optimal stimulation results were obtained using the following conditions: PHA-M in a dilution of 1:15, fetal calf serum (FCS) in a concentration of 4%-10%, a cell concentration of 4 X 10(6) - 1.5 X 10(7) cells/ml, RPMI 1640 Medium or McCoy's 5A Medium, an incubation period of 48 h - 72 h at a temperature of 39 degrees C, and low-speed centrifugation of heparinized blood for lymphocyte collection. The findings of the present study are discussed in the context of results described in the literature.