SEPARATION OF EOSINOPHILS BY DIFFERENTIAL PERCOLL CENTRIFUGATION

Abstract

Helminth infections are known to cause an increase in the number of eosinophils in peripheral blood, and there is evidence that these "activated" eosinophils are less dense than normal eosinophils. In order to study changes in swine eosinophil populations in response to a Trichinella spiralis challenge infection, it was necessary to refine and standardize a protocol for separating eosinophils from whole blood. Discontinuous Percoll gradient centrifugation was the separation method chosen. The revised protocol is as follows: collect whole venous blood in sodium citrate, direct count the eosinophils and dilute the sample to 500 eo/ul with lx HBSS without Ca⁺⁺ or Mg. Dextran sediment the blood at room temperature with 5% dextran T 500 which is 4 ° C when mixed with the blood, 1.1 m1:10 ml sample. Sedimentation should begin within 30 min of blood drawing, and the optimum sedimentation time is 15 min. It is important to completely harvest the supernatant by pipetting, even if this results in contamination by some erythrocytes from the underlying layer. Wash the supernatant and dilute it with lx HBSS without Ca ⁺⁺ or Mg⁺⁺ with EDTA, and centrifuge for 8 min at 300 g, 20 ° C, no brake. Resuspend the pellet in HBSS without Ca ⁺⁺ or Mg⁺⁺ plus peripheral blood lymphocyte (PBL) preparation medium (HBSS without Ca ⁺⁺ or Mg, 0.01M EDTA, 0.01M Hepes) and overlayer onto a Percoll gradient (54%-65%-75%-85% Percoll). Centrifuge the gradient for 25 min at 475 g, 200 C, no brake. Wash the harvested layers with HBSS without Ca ⁺⁺ or Mg⁺⁺ and bring up the final pellet in 1 ml serum-containing medium (5% newborn calf serum <Sigma: N-0515>) for counting. In this study, responses of different swine leukocyte antigen (SLA) genotypes were also compared with respect to differences in the eosinophilic response, but results were inconclusive, and that aspect of the study is ongoing.