Separation and phagocytosis analysis of peripheral blood leukocytes from Qihe crucian carp Carassius auratus.

The phagocytic function of peripheral blood leukocytes (PBL) in teleost fish is the bridge between cellular-mediated and humoral immunity and plays a central role in the immune system of the teleost fish. However, the phagocytic ability of PBL from Qihe crucian carp Carassius auratus remains unclear. This study aimed to build a convenient separation method to obtain high purity PBL from Qihe crucian carp and to explore their phagocytic characteristics. The results showed that the PBL of Qihe crucian carp (QPBL) could be obtained effectively by 1.085 g/ml percoll separation liquid. Most isolated QPBL were round or elliptical, with horseshoe-shaped, reniform, or irregular nucleus. Finger-like or filamentous protrusions were present on the cell surface, and some cells had a long pseudopod. According to the results of scanning electron microscopy (SEM), transmission electron microscopy (TEM), and fluorescence microscope, fluoresbrite carboxylate YG microspheres (1 μm) could be effectively phagocytosed by QPBL, and various phagocytic stages could be observed. The phagocytic cells in QPBL have morphology similar to those of "professional" phagocytes (monocyte and granulocyte). Notably, the percentage of cells, which can engulf microspheres, increased to approximately 22% after incubation with microspheres for 90 min, and it showed no obvious changes until 180 min. Furthermore, two important phagocytosis regulatory protein genes, Rab5 and Rab7 , were seriously up-regulated during the phagocytosis in QPBL. Taken together, an efficient and convenient separation method for isolating QPBL was established, and the isolated QPBL had a high phagocytic activity towards the external particles. The results could provide the basic foundation for further study of the immune function of PBL in Qihe crucian carp and other teleost fish. • The PBL of Qihe crucian carp (QPBL) could be separated by 1.085 g/ml percoll separation liquid. • The QPBL had a high phagocytic activity towards the external particles. • The proportion of phagocytes in QPBL was about 22%. • Rab5 and Rab7 were involved in the phagocytic activity of QPBL. [ABSTRACT FROM AUTHOR]