Optimum conditions for the turkey lymphocyte transformation test

Optimum conditions for turkey lymphocyte transformation tests were determined. Thrice-washed turkey buffy-coat cells obtained after slow centrifugation (40 X g, 10 minutes) responded well to mitogenic stimulation. Turkey lymphocytes isolated on Ficoll-containing separation media largely lost their ability to respond to mitogens. Maximum responses were obtained with 2 X 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when bovine fetal serum was used at a 2.5% concentration or pooled turkey serum and autologous plasma were used at a 1.25% concentration. Higher concentrations of turkey serum or plasma decreased the responses when sub-optimum doses of concanavalin-A (Con A) or phytohemagglutinin-P (PHA-P) were used. Serum free cultures gave higher stimulation indices than cultures with serum only when sub-optimum doses of Con A or PHA-P were used. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. Responses were usually greatest with final concentrations of 5 microgram Con A/ml, 10 microgram PHA-P/Ml, and 20 microgram pokeweed mitogen (PWM)/ml and when the cultures were incubated in 96-well microplates at 40 degrees C in humidified air with 5% CO2 for 40-42 hours with pulsing with 3H-thymidine during the final 16 hours of incubation