Analysis of Salmonid Leukocytes Purified by Hypotonic Lysis of Erythrocytes

Abstract

A technique that uses hypotonic lysis of erythrocytes was optimized for the purification of leukocytes from the peripheral blood and anterior kidney (pronephros) of rainbow trout Oncorhynchus mykiss. Comparisons of initial blood dilution (1:2, 1:4, and 1:6) and the time of exposure to hypotonic conditions (10, 20, and 40 s) revealed that a dilution of 1:2 provided the most complete hemolysis after 20 or 40 s in a hypotonic solution. For pronephros, a 1:5 (w:v) dilution and lysis in hypotonic solution for 10–40 s was effective in eliminating erythrocytes. Total leukocyte yield from the blood and pronephros by use of the hypotonic lysis method was comparable with that obtained by use of typical density gradient centrifugation, and cell viability was 97% or greater. Differential cell counts showed that hypotonic lysis resulted in a distribution of leukocyte cell types similar to that of density gradient separation. Hypotonic lysis of erythrocytes is a simple, rapid, and inexpensive method of purifying leukocytes from salmonid fish blood and pronephros.

Notes

² The large-blood-volume protocol was as follows: 2.0 mL of blood was diluted with 2 mL of isolation medium and placed into a 50-mL centrifuge tube. An aliquot of 36 mL of dH2O was added, and the cell suspension was mixed for 20 s. Immediately after mixing, 4 mL of 10× PBS was added and mixed thoroughly. The analysis continued as described in the small-volume protocol.