Is the In Vitro Observed NETosis the Favored Physiological Death of Neutrophils or Mainly Induced by an Isolation Bias?

Abstract

Centrifugation steps are regularly used for neutrophil isolation. Thereby, the influences of applied g-forces on the functionality of PMNs have hardly been analyzed and could consequently have been overlooked or led to biased results. We now hypothesize that blood PMNs-when gently isolated-can be long-lived cells and they physiologically become apoptotic rather than NETotic. Neutrophils were isolated from whole blood without centrifugation using a sedimentation enhancer (gelafundin). PMNs were analyzed via live-cell imaging for migratory activity and vitality condition by fluorescent staining. Native neutrophils showed still relevant migratory activity after more than 6 days ex vivo. The percentage of cells that were annexin V+ or PI+ increased successively with increasing ex vivo time. In addition, the characteristics of DAPI staining of gently isolated granulocytes differed markedly from those obtained by density gradient separation (DGS). We conclude that NETosis occurring after DGS is the consequence of applied g-forces and not a physiological phenomenon. Future studies on neutrophils should be performed with most native cells (applied g-time load as low as possible).