The Application of Dextran Sedimentation as an Initial Step in Neutrophil Purification Promotes Their Stimulation, due to the Presence of Monocytes

Abstract

The purification of human neutrophils for in vitro studies is challenging as they are easily activated through ex vivo manipulations. The technique of erythrocyte sedimentation combined with density gradient centrifugation remains widely practiced and was the subject of this study. Since in the sedimentation step the leukocytes are incubated with dextran, we have raised the likelihood that cellular activation would occur with mediator release leading to neutrophil activation. By comparing the activity of neutrophils purified from whole blood by the classical 2-step method of dextran sedimentation followed by low-density Ficoll-Hypaque (1.077 g/mL) medium, and the 1-step high-density Ficoll-Hypaque (1.114 g/mL) gradient centrifugation, we found that neutrophils from the 2-step method had a significant increase in cell surface CD11b expression and CD62L shedding and a marked increase in adhesion. Decreased random migration and chemotaxis and raised baseline oxidative burst activity were also observed. The effect was not specific to dextran, as using Ficoll for erythrocyte sedimentation replicated the elevated neutrophil adherence. Through the depletion of monocytes, lymphocytes, and platelets prior to sedimentation, we deduced that monocytes were responsible for the neutrophil activation. Our findings suggest that care needs to be exercised in choosing the method of neutrophil purification for functional studies.

AIDeveloper: deep learning image classification in life science and beyond

“…RT-DC is an imaging flow cytometer where bright-field images of cells in flow are captured by a high-speed camera ( Figure 3A) at rates of 100 -1,000 cells/s. In order to increase the frequency of leucocytes, whole blood was first depleted from RBCs by dextransedimentation (Bøyum, 2008;Quach and Ferrante, 2017). As recently published by Toepfner et al, the individual cell populations can be identified simply by considering cross-sectional area and brightness, calculated as the average grayscale value of all pixels belonging to the cell ( Figure 3B).…”

“…To prepare RBC-depleted blood samples, 2 mL of a 6% dextran solution (Dextran T500, Pharmacosmos A/S, Denmark) diluted in sodium chloride (0.9% Sodium Chloride Irrigation, Baxter Healthcare, Switzerland) was added to 10 mL of whole citrated blood. After gentle mixing, RBCs were allowed to sediment for 30 min (Bøyum, 2008;Quach and Ferrante, 2017). The supernatant was transferred to a 10 mL tube and centrifuged for 10 min at 120 g (Universal 30RF, Hettich, Switzerland).…”

Calcium signaling and regulation of neutrophil functions: Still a long way to go

“…We also need to keep in mind that the pathways engaged in NETosis can be influenced by the state of priming and pre-activation of neutrophils due to the techniques of purification. 124,125…”

The Role of Phagocytes in Immunity to Candida albicans

“…Similar to macrophage phagocytosis of C. albicans, uptake of isolated fungal zymosan A is more efficient in opsonising conditions, with phagocytosis after a 15-min incubation time being three times higher in reactions with complement compared to no serum and heat-inactivated serum controls (Figure 1). were prepared from human peripheral blood from healthy volunteers, using the high density gradient method [17].…”