Optimum conditions for the chicken lymphocyte transformation test.
Optimum conditions for chicken (Gallus gallus) lymphocyte transformation tests were determined. Thrice-washed chicken buffy-coat cells obtained after slow centrifugation (40 x g for 10 minutes) responded substantially better to mitogenic stimulation than lymphocytes isolated on separation media containing Ficoll. Maximum responses were obtained with 2 x 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when fetal bovine serum was used at a 5% concentration or pooled chicken serum and autologous plasma were used at a 1.25% concentration. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. When 1.25% chicken serum was used in the cultures, responses were usually greatest with final concentrations of 30-50 micrograms/ml of concanavalin A (Con A) and 30-50 micrograms/ml of phytohemagglutinin-P (PHA-P). The optimum concentration of pokeweed mitogen (PWM) varied from 1 to 40 micrograms/ml among the birds and was practically impossible to establish in general. The incubation in humidified air with 5% CO2 was significantly better at 40 C than at 37 C. The total culture time of 40 hours including pulsing with 3H-thymidine during the final 16 hours of incubation was the best for Con A- and PHA-P-stimulated cells, whereas a longer incubation of 64 hours gave the highest results with PWM stimulations.
Barta
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Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg 24061.
Barta
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Pierson
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D000818:Animals; D001769:Blood; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D002645:Chickens / Q000276:immunology*; D003470:Culture Media; D003973:Diatrizoate; D004912:Erythrocytes; D005362:Ficoll; D008213:Lymphocyte Activation; D013696:Temperature; D013997:Time Factors
D003470:Culture Media; D003973:Diatrizoate; D005362:Ficoll