DETECTION OF CANINE INTERLEUKIN-2 RECEPTORS BY FLOW-CYTOMETRY

This study describes a method for detecting canine interleukin-2 receptors (IL-2R) by flow cytometry, using human recombinant IL-2 labeled with phycoerythrin (IL-2-PE). Peripheral blood mononuclear cells from four normal dogs were washed, incubated with IL-2-PE, and then washed to remove any unbound IL-2-PE. Flow cytometric analysis of the cells was performed with a 488 nm argon laser while gating on lymphocytes. Cells expressing the IL-2R were identified by their fluorescence as compared to cells stained with an anti-mouse immunoglobulin-G conjugated to phycoerythrin. The average percentage of resting cells expressing the IL-2R was found to be 21%. The addition of unlabeled human recombinant IL-2 to Day 3 phytohemagglutinin (PHA)-stimulated cells reduced the fluorescence intensity four-fold, thereby demonstrating the specificity of IL-2-PE for canine IL-2R. Following stimulation with optimal concentrations of PHA, the percentage of cells expressing the IL-2R increased daily and reached a maximum on Day 3 (76.4%). IL-2R density, as measured by mean fluorescence intensity, also increased and reached maximal levels on Days 2-3 (twenty-fold greater than resting cells). The binding, inhibition, and kinetic experiments provide evidence that human recombinant IL-2-PE is a useful tool for studying canine IL-2R expression. Thus, a one-step direct method for the flow cytometric detection and quantification of the canine IL-2R is now available.