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Published on: September 17, 2021
Separation methods for isolation of human polymorphonuclear leukocytes affect their motile activity
Jolanta Sroka1, Anna Kordecka, Przemysław Włosiak
1Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow, Poland.
Abstract:
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.
Insights
Comparing five human polymorphonuclear leukocyte (PMNL) isolation methods, researchers found that blood clot and Percoll methods best preserve PMNL motile activity. All methods maintained cell viability and receptor sensitivity for chemotaxis.
Area of Science:
- Immunology
- Cell Biology
Background:
- Human polymorphonuclear leukocytes (PMNLs) are crucial immune cells.
- Effective isolation methods are vital for studying PMNL function.
- Previous studies have not comprehensively compared PMNL isolation techniques' impact on motility.
Purpose of the Study:
- To compare five common human PMNL isolation methods.
- To assess the effects of these isolation methods on PMNL motile activity.
- To evaluate PMNL receptor sensitivity post-isolation.
Main Methods:
- Isolation of human PMNLs using five distinct methods: blood clots, hypotonic hemolysis, Percoll, Ficoll 400, and Dextran T 500.
- Assessment of cell viability, spontaneous cell movement, and N-FMLP-mediated chemotaxis.
- Immunofluorescence microscopy to observe cell surface characteristics.
Main Results:
- All isolation methods preserved PMNL viability.
- Blood clot and Percoll methods yielded the highest PMNL motile activity.
- While N-FMLP stimulation enhanced movement across all methods, Dextran T 500 and Ficoll 400 isolations resulted in relatively slower cell movement.
- Immunofluorescence revealed FITC-Dextran coated PMNLs without disrupting receptor accessibility.
Conclusions:
- The choice of isolation method significantly impacts PMNL motile activity.
- Percoll and blood clot methods are optimal for preserving PMNL motility.
- PMNLs retain chemotactic receptor sensitivity regardless of the isolation method used.
- Polysaccharide coating during isolation does not impede flow cytometry analysis.
