Isolation of Leucocytes from Fish Blood

Abstract

Procedures for isolating of the leucocytes from fish blood were studied. Blood samples were obtained by heart puncture from carp, Cyprinus carpio and gold fish, Carassius auratus. The following two methods were adopted.
Method 1. To one volume of the blood collected into a heparinized plastic syringe, added 2/3 volumes of physiological saline for freshwater fish(PSF). Mixed well by inversion. Vertically kept the syringe at room temperature for 3 hours. Collected the leucocytes from the buffy coat.
Method 2. To heparinized blood, added an equal volume of PSF. Layered the blood-saline mixture onto the ficoll and sodium diatrizoate mixture(Histopaque®). Centrifuged at 400×g for 30 minutes at room temperature. Collected the. leucocytes from the opaque interphase.
About 10⁶ cells of leucocytes were isolated from 1 ml of the blood by these methods. The viability of isolated leucocytes was shown by their non-staining with trypan blue and phagocytic ability on bacterial cells. The peroxidase negative leucocytes had the adhesive property to the glass surface. These results indicate that the leucocytes isolated by these two methods are useful for studying the functions of fish leucocytes in vitro.