Centrifugation is a common step in most of the popular protocols for the isolation of neutrophils from whole blood. Inconsistent results from previous studies on neutrophils may originate from an underestimation of the centrifugation effect, as in consequence impaired, not native cells, being investigated. We hypothesize, that centrifugation significantly impairs major neutrophil functions. However, there is no data yet whether the application of g-force itself or the product of g-force and duration of centrifugation (=“g-time”) defines the impact on neutrophils. Neutrophils were isolated from whole blood via centrifugation with different g-times and subsequently analyzed via live cell imaging for migration, as well as via flow cytometry for oxidative burst and surface antigen expression. Chemotactic migration was significantly reduced with increasing g-time. Oxidative burst decreased likewise the higher the g-time applied. Expression of CD11b was no longer upregulated in response to an n-formylmethionine-leucyl-phenylalanine (fMLP) stimulus in neutrophils having experienced high g-time during the isolation process. We conclude that centrifugation “paralyzes” neutrophils in the form of a significant decrease in functionality. Future investigations on neutrophil granulocytes should reduce the g-time load as far as possible.

1. N-formyl peptides (e.g. fMLP; N-formyl-L-methionyl-L-leucyl-phenylalanine) are potent mediators for inflammatory reactions. We report functional expression in Xenopus oocytes of human fMLP-R98 cDNA, without co-expression of the promiscuous G-protein subunit, Galpha-16. 2. Stimulation of voltage-clamped oocytes (-70 mV) with fMLP produced a dose-dependent biphasic inward current with fast and slow components. Analysis using GTP-gamma-S and cholera and pertussis toxins suggested these currents are mediated by an endogenous G-protein of the Gq family. 3. The fast current reversed at -25 mV and was blocked by SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid), suggesting the current is carried by Cl(-). The slow current showed weak inward rectification, was Ca(2+)-dependent and blocked by Cd(2+), 4-AP (4-aminopyridine) and haloperidol, suggesting activation of a mixed population of cation channels. 4. Comparative experiments with human neutrophils using flow cytometric analysis showed that the proportion of neutrophils activated by fMLP was reduced in the presence of SITS, in the absence of external calcium and in the presence of Cd(2+), TEA (tetraethylammonium) and haloperidol but not 4-AP. In addition, the oxidative burst from activated neutrophils was reduced by SITS and by the absence of external calcium but not by Cd(2+), TEA, 4-AP or haloperidol. 5. We suggest that in human neutrophils activation by fMLP is dependent on store-operated calcium influx that appears to be regulated by Cl(-) channels and linked, in part, to non-selective cation channels.

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).

To elucidate potential stereoselective effects of single barbiturate isomers, we compared the inhibitory potency of single thiopentone enantiomers, two isomer-enriched mixtures of methohexitone and racemic mixtures of both barbiturates on the fMLP-induced neutrophil oxidative response. A suppression of the response to 50% compared to control required a 100-fold therapeutic concentration of methohexitone, while therapeutic concentrations of the thiopentone racemate led to a significant inhibition (relative fluorescence of neutrophils 0.46 ± 0.03 compared to fMLP controls). The racemate of thiopentone produced significantly greater inhibition than the single enantiomers. Stereoselectivity in favor of one isomer could not be shown for both barbiturates. The greater inhibition by the thiopentone racemate might suggest two separate binding sites for the enantiomers which are positively coupled.

To investigate granulocytes under laboratory conditions, centrifugation steps are typically required for the isolation of neutrophil granulocytes from whole blood. However, only a few studies to date have addressed the direct effects of centrifugation itself on the functional state of neutrophils. This study aims to elucidate the mechanisms that contribute to the modification of granulocytes during centrifugation. We hypothesize that granules sustain morphological alterations during centrifugation, leading to the release of highly potent antimicrobial enzymes into the cytosol of the cells. Neutrophils were isolated from whole blood using different methods with and without centrifugation and analyzed by flow cytometry, ELISA, and mass spectrometry. Our findings demonstrate that intracellular granules incur damage during centrifugation, resulting in the presence of intragranular enzymes within the cytosol. Furthermore, the formation of the highly reactive hypochlorous acid (HOCl) as a consequence of centrifugation could be verified. The generation of intracellular HOCl may explain many of the alterations observed in neutrophils following centrifugation-based isolation, including modified surface antigen expression and altered responses to stimulation. In future studies, centrifugation steps during cell isolation should be avoided. The more time-consuming but gentler method of sedimentation is preferable and can be used as long as it is not necessary to obtain a highly purified neutrophil fraction.