The Application of Dextran Sedimentation as an Initial Step in Neutrophil Purification Promotes Their Stimulation, due to the Presence of Monocytes
The depletion of monocytes from blood decreases adhesion modulation during dextran sedimentation. Configurations of “reconstituted” blood was applied to the 2-step method with dextran sedimentation preceding low-density Ficoll-Hypaque gradient centrifugation. (a) Schematic flow chart of the process for preparing configurations of “reconstituted” blood and the purification of neutrophils. (b) Column graph of adherence of neutrophils purified from the various configurations of blood with (+), without (−), and/or depletion (dep.) of platelets and PBMCs. (c) Graph of percentage changes from reconstituted blood with PBMC (PBMC (+)) of CD14+ monocyte % of PBMC as a result of depletion prior to application of reconstituted blood onto the 2-step method, and corresponding CD18, CD11b, and CD62L MFI of neutrophils following purification (). (d) Representative flow cytometry plot of CD14+ monocytes pre- and post-depletion from PBMCs by adhesion to fibronectin-coated polypropylene; representative histogram offset overlays of the fluorescence intensities of neutrophil surface CD11b and CD62L expression between the blood configurations. (e) TNF concentrations in the plasma extracted from blood (not applied to dextran sedimentation) and the supernatant of centrifuged leukocyte-enriched plasma immediately following dextran sedimentation ().
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