Vet Med - Czech, 2014, 59(11):543-552 | DOI: 10.17221/7819-VETMED

The impact of pre-analytical treatment and sorting on human neutrophil functionOriginal Paper

L. Hromadkova¹, L. Zarybnicka², J. Vavrova², Z. Bilkova¹, Z. Sinkorova²

¹ University of Pardubice, Pardubice, Czech Republic

² Faculty of Military Health Sciences, University of Defence Brno, Hradec Kralove, Czech Republic

This study was designed to test the hypothesis that pre-analytical treatment of relatively fragile cells is more important for their viability and function than shear forces and decompression shocks on a flow cytometric sorter. Human neutrophils were enriched using different techniques (sedimentation, hypotonic or ammonium chloride-mediated erythrocyte lysis) and their function was evaluated by measuring their capability of mounting oxidative burst. In contrast to other isolation techniques, neutrophils enriched by spontaneous sedimentation were found to be intact both in terms of their function and relative numbers within the leukocyte population; such preparations were thus run on a FACSAria cell sorter using 70, 85 and 100 µm nozzles and the corresponding internal pressure values. The viability and function of sorted neutrophils with or without subsequent cultivation were re-evaluated using phagocytosis assays. Independent of the nozzle size and internal sorter pressure, the capability of neutrophils of responding to E. coli stimulation was impaired after sorting while phorbol myristate acetate stimulation remained intact. In tested samples, only 20 h incubation of sorted cells confirmed the expected influence of the nozzle size on the sorted cell function. We conclude that pre-analytical treatment is more important for cell function than conditions during sorting.

Keywords: flow cytometry; cell sorting; granulocytes; phagocytosis; oxidative burst

Published: November 30, 2014  Show citation