Culture methods for turtle lymphocytes.
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 microg/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.
Ulsh
B A
BA
Department of Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523, USA.
Congdon
J D
JD
Hinton
T G
TG
Whicker
F W
FW
Bedford
J S
JS
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002875:Chromosomes / Q000187:drug effects / Q000502:physiology; D003470:Culture Media; D007376:Interleukin-2 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008214:Lymphocytes / Q000097:blood / Q000166:cytology* / Q000187:drug effects; D008387:Marine Toxins; D008938:Mitosis / Q000187:drug effects / Q000502:physiology*; D010080:Oxazoles / Q000494:pharmacology; D010835:Phytohemagglutinins / Q000494:pharmacology; D013997:Time Factors; D014426:Turtles / Q000097:blood*
D003470:Culture Media; D007376:Interleukin-2; D008070:Lipopolysaccharides; D008387:Marine Toxins; D010080:Oxazoles; D010835:Phytohemagglutinins; C047876:phytohemagglutinin-M; C059041:calyculin A