A Technique for Separation of Canine Lymphocytes and Their Use in the Lymphocytotoxic, Blastogenic, and Rosette Assays

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Abstract
When the techniques established for preparing lymphocytes from human blood were applied to canine blood, the resulting preparations were contaminated with significant numbers of other blood cells. A three‐step technique is described here for the separation of canine lymphocytes from blood which eliminates most granulocytes, platelets, and erythrocytes. Defibrinated blood is incubated with iron particles to allow phagocytosis by granulocytes. When mixed with Plasmagel, erythrocytes and iron‐containing granulocytes sediment rapidly, leaving a lymphocyte‐rich supernate plasma which is recovered and banded by centrifugation on a Ficoll‐Hypaque gradient. Any erythrocytes remaining in the lymphocyte suspension aspirated from the gradient are eliminated by hypotonic lysis and centrifugation. The resulting preparation, representing 30 per cent recovery of the circulating lymphocytes, contained 85 to 94 per cent lymphocytes with a cell viability of 99 per cent. The functional capacity of the lymphocyte preparation was tested in three assays: 1) lymphoblastic transformation using phytohemagglutinin and pokeweed mitogen, 2) E rosette formation with human erythrocytes showed a mean 5 per cent rosette forming lymphocytes, 3) antibody‐mediated lymphocytotoxicity using a canine anti‐lymphocyte serum showed titers reproducible within one tube dilution.