Isolation and characterization of macrophages from goldfish (Carassius auratus) head kidney and spleen by density-gradient separation

Fish like other vertebrates, respond to infectious agents in both specific and nonspecificmanner, and appear to have similar cellular and humoral immunecomponents as warm blooded vertebrates. Macrophages are important cells indiseases resistance of fish. They are the main phagocytic cells in fish and alsoparticipate in specific immune response by processing and presenting antigens. Togain a better of the host s defence mechanisms, it is necessary to establish methods tostudy the cells involved in defence reaction under controlled condition. In the presentstudy, spleen and head kidney macrophages from goldfish were isolated by Percolldensity-gradient separation. The spleen and head kidney were aseptically removedand pushed through a nylon mesh with specially formulated medium Dulbecco sModified Eagle Medium (DMEM). After centrifugation, the cells was collected andwashed. For identification of cells, air-dried smears of the different samples werestained with Haemacolor® stain and observed under the light microscope. Thepresence of non-specific esterase activity was also determined using a special stainingkit. The average proportion of esterase positive cells (macrophages) wasapproximately 90% in cell suspension. The cells were counted and their viability wasdetermined. The amount of adherent cells was also determined after incubation at 22°C. The adhering cells per microscopic field were counted. In conclusion,approximately 2 x 10 6 macrophages from each head kidney and 5 x 10 5 macrophagesfrom each spleen were harvested. The viability exceeded 95% but after 24-48 hoursviability decreased to 10 -15%, significantly.

Keywords:

Macrophages , Goldfish , Head kidney , Spleen