title: Simple method for culture of  peripheral blood lymphocytes of Testudinidae.
abstract: We  developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis  carbonaria (Testudinidae), testing various parameters, including culture  medium, mitogen concentration, mitotic index, culture volume, incubation time,  and mitotic arrest. Peripheral blood samples were obtained from the costal vein  of four couples. The conditions that gave a good mitotic index were lymphocytes  cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin  as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an  incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to  colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest,  the cells were hypotonized with 0.075 M KCl for 2 h and fixed with  methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized  by Giemsa staining. The diploid chromosome number of C. carbonaria was  found to be 52 in females and males, and sex chromosomes were not observed. We  were able to culture peripheral blood lymphocytes of a Brazilian tortoise in  vitro, for the preparation of mitotic chromosomes.
authors.affiliation: Departamento de Biologia, Centro de Estudos de Quelônios, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista "Júlio de Mesquita Filho", São José do Rio Preto, SP, Brasil. lucenabio@hotmail.com
authors.forename: T L
authors.initials: TL
authors.lastname: Silva
authors.forename: M I A
authors.initials: MI
authors.lastname: Silva
authors.forename: L P R
authors.initials: LP
authors.lastname: Venancio
authors.forename: C E S
authors.initials: CE
authors.lastname: Zago
authors.forename: V A G
authors.initials: VA
authors.lastname: Moscheta
authors.forename: A V B
authors.initials: AV
authors.lastname: Lima
authors.forename: L D
authors.initials: LD
authors.lastname: Vizotto
authors.forename: J R
authors.initials: JR
authors.lastname: Santos
authors.forename: C R
authors.initials: CR
authors.lastname: Bonini-Domingos
authors.forename: M T V
authors.initials: MT
authors.lastname: Azeredo-Oliveira
mesh_terms: D000818:Animals; D001399:Azure Stains / Q000032:analysis; D001938:Brazil; D018929:Cell Culture Techniques; D002871:Chromosome Banding; D002875:Chromosomes / Q000737:chemistry / Q000235:genetics*; D003078:Colchicine / Q000494:pharmacology; D003470:Culture Media / Q000737:chemistry*; D005260:Female; D005404:Fixatives; D006801:Humans; D008214:Lymphocytes / Q000166:cytology* / Q000187:drug effects; D008297:Male; D008934:Mitogens / Q000494:pharmacology; D008940:Mitotic Index; D010835:Phytohemagglutinins / Q000494:pharmacology; D014426:Turtles / Q000235:genetics*
chemical_list: D001399:Azure Stains; D003470:Culture Media; D005404:Fixatives; D008934:Mitogens; D010835:Phytohemagglutinins; D003078:Colchicine