Cellular function evaluation of sanguineous leukocytes in Phrynops geoffroanus using flow cytometric methodology

Although Phrynops geoffroanus displays ample geographical distribution in South America, few studies had been developed in relation to the natural history of this freshwater turtle. This species frequently appears in contaminated water sources, but the effect of adverse situations (anthrophic effects) on the animals' cellular immune activity is unknown. In thiswork amethod employing flow cytometry was developed in order to evaluate the cellular functions of phagocytosis and spontaneous and induced oxidative burst in leukocytes from blood samples of Phrynops geoffroanus found in contaminated environments of the Piracicaba river and Piracicamirim brook, in São Paulo State. Blood samples were obtained from 8 animals from Piracicaba river, 9 from Piracicamirim brook, and 7 from Zoological Park Foundation of São Paulo; the samples from zoological park were used as control group. The samples had been kept in culture in RPMI medium under refrigeration and directed to the Neuro-immunology Laboratory of Department of Pathology in FMVZ/USP to be processed. The fact that the red blood cells are nucleated in these animals, made necessary the use of Ficoll-PaqueTM PLUS at different densities (1085, 1070 and 1055 g/mL), in order to pre-separate these cells. Then the sample was centrifuged to obtain cells of interest, at 18 oC, 450ÿg, acceleration 9 and brake 0, resulting in a white ring of leukocytes located between the superior layers of the Ficoll. The samples were submitted to twowashes with RPMI to remove the excesses of Ficoll. After the washes, the samples were suspended again in 500 ML RPMI for making a viability counting with Trypan Blue; samples with inferior percentages to 94 % were not used in the stimulates: Zymosan A (Sacaromyces cerevisiae) the Bio Particles®, Alexa Fluor® 594 conjugate to induce phagocytosis and 12-myristate 13-acetic acid (PMA) and Sacaromyces cerevisiae (Zymosan) in the induction of oxidative burst. The animal's leukocytes of the zoo presented greater phagocytosis intensity induced by Zymosan A, in relation with to the cells of the animals of the Piracicaba river. The intensity of oxidative burst induced by PMA was smaller in the animals of the Piracicamirim brook when compared with turtles of Piracicaba river and the zoo.