The primary culture of carp (Cyprinus carpio) macrophages and the verification of its phagocytosis activity

This study establishes the primary culture method for red carp (Cyprinus carpio) macrophages in vitro and lays the foundation for further research in the fish immune system. The healthy adult red carp was chosen, and mechanical separation and cell adherent culture methods were used to isolate the primary macrophages. Compared to the traditional method of Percoll discontinuous density gradient isolation, the protocol we reported here makes cell isolation steps more concise and obtains more healthy cells with high macrophage purity. The cells were uniform in size with a clearly visible nucleus. Trypan blue staining and non-radioactive cell proliferation assay were used to detect the cell survival rate. Further, we provide optimum culture conditions which include cell density (1 × 10(7) cells/mL), culture medium (Leibovitz's L-15), pH (7.2-7.4), temperature (26°C), and adherent time (24 h). Macrophages have been identified by nonspecific esterase and Wright-Giemsa staining and have shown to grow very well. In addition, the macrophages have a very strong bactericidal activity against three kinds of bacteria, further verifying good growth conditions and proper function.