Analysis of Salmonid Leukocytes Purified by Hypotonic Lysis of Erythrocytes

—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. Interest in the study of functional teleost immunology necessitated the development of a method to rapidly separate leukocytes from erythrocytes. Typically, teleost leukocytes from peripheral blood or tissues are purified by continuous or discontinuous density gradient centrifugation through separation media such as Histopaque, Ficoll-Paque, or Percoll (Blaxhall 1981; Blaxhall and Sheard 1985; Braun-Nesje et al. 1981; Huffman et al. 1997). Density gradient preparations are * Corresponding author: [email protected] 1 Present address: Agricultural Research Service, Southern Plains Agricultural Research Center, U.S. Department of Agriculture, 2881 F&B Road, College Station, Texas 77845, USA. Received December 4, 2000; accepted April 23, 2001. tedious, time consuming, and expensive and may cause unwanted stimulation of immune cells. The use of Ficoll has been shown to alter morphology and function in heterophils, as well as to activate some leukocyte subsets (Ogle et al. 1985; Verhoef and Waldvogel 1985). The separation of pure, viable, and quiescent leukocytes is essential for subsequent use of cells in in vitro immunological as-