SREL Reprint #2561

Culture methods for turtle lymphocytes

B. A. Ulsh1, J. D. Congdon2, T. G. Hinton2, F. W. Whicker1, and J. S. Bedford1

1Department of Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523, USA
2Savannah River Ecology Laboratory, Drawer E, Aiken, SC 29802, USA

Abstract: Optimization of culture techniques for turtle and other reptilian lymphocytes is essential for facilitating cytogenetic and immunologic research for these animals. We examined a variety of conditions and parameters relevant to turtle lymphocyte culture including: different mitogenic agents, alone and in combination; lymphocyte separation protocols; culture volume; time required to stimulate lymphocytes to mitosis; importance of humidity and gas exchange in culture incubation; suitability of different culture media; effects of varying serum concentrations; ability of interleukin-2 (IL-2) to stimulate lymphocyte growth and prevent apoptosis; and feasibility of inducing premature chromosome condensation. The best conditions of those we studied for obtaining mitotic cells were (1) the combined use of phytohemagglutinin-M form (2%) and lipopolysaccharides (0.55 µg/ml), (2) the use of 5% autologous turtle serum (as opposed to fetal bovine serum), and (3) collection of mitotic cells around 96 hours after mitogenic stimulation. Human, recombinant IL-2 did not increase the fraction of lymphocytes in mitosis over the range of concentrations tested and calyculin A was ineffective at inducing premature chromosome condensation in turtle lymphocytes over the range of concentrations tested. This test regime provides a guideline for determination of appropriate lymphocyte culture conditions in turtles and other reptiles.

Keywords: Blood culture, Lymphocyte stimulation, Metaphase accumulation, Reptile, Turtle

SREL Reprint #2561

Ulsh, B. A., J. D. Congdon, T. G. Hinton, F. W. Whicker, and J. S. Bedford. 2001. Culture methods for turtle lymphocytes. Methods in Cell Science 22:285-297.

This information was provided by the University of Georgia's Savannah River Ecology Laboratory (srel.uga.edu).