Separation methods for isolation of human polymorphonuclear leukocytes affect their motile activity

Five commonly used methods for the isolation of human polymorphonuclear leukocytes (PMNLs) from blood and their subsequent effect on cell motile activity were compared. Although all methods (isolation from blood clots, hemolysis of erythrocytes in hypotonic solutions, and sedimentation with the use of Percoll, Ficoll 400 or Dextran T 500 solutions) preserved cell viability, they demonstrated different effects on cell spreading and the speed of spontaneous cell movement. The highest motile activity was shown by PMNLs separated from blood clots and Percoll solutions. In the presence of formylated peptides, N-FMLP-mediated movement was markedly stimulated in PMNLs separated by all five methods, but cells isolated with the use of Dextran T 500 or Ficoll 400 were relatively slower than those isolated with other methods. This suggests that the cells had preserved the sensitivity of their receptors specific for ligands stimulating chemokinesis (chemotaxis) after all five methods of separation. Immunofluorescence observations showed that PMNLs isolated in the presence of FITC-Dextran exhibited polysaccharide-coated surfaces with receptor proteins extended above that coat – an observation which explains why cell coating with polysaccharides does not disturb cell phenotyping with flow cytometry and FACS methods.