Identification of the Centrifugation-Caused Paralytic Impact on Neutrophils

Abstract

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.

Keywords: HOCl; Myeloperoxidase; centrifugation; granules; impairment; isolation; neutrophil functions; neutrophils; reactive oxygen species; surface antigen expression.

Figure 1

Parameters analyzed to assess the effects of centrifugation on PMNs: granule integrity (intra- and extracellular enzyme levels), formation of HOCl from H₂O₂ (quantified as 3-chlorotyrosine), and PMN functional state (oxidative burst and surface antigen expression).

Figure 2

Shown are EC₅₀ (A,C,E) and E_max (B,D,F) values for MPO (A,B), lactoferrin (C,D), and MMP9 (E,F) obtained from different experiments comparing sedimented and centrifuged PMNs.

Figure 3

Extracellular MPO concentration [pg/mL] at different digitonin concentrations [µg/mL] in sedimented and centrifuged PMNs (A); relative change in extracellular MPO concentration in centrifuged PMNs compared to sedimented PMNs (B); mean values and standard deviations of the relative changes in extracellular MPO concentration when digitonin is present (C).

Figure 4

Median fluorescence intensities [AFUs] of secondary anti-rabbit IgG binding to the primary antibody against 3-chlorotyrosine in centrifuged versus sedimented PMNs at different digitonin concentrations [µg/mL].

Figure 5

Quantification of the oxidative burst by measuring median rhodamine 123 fluorescence intensity [AFU] after different stimulations (unstimulated, fMLP + TNF, and PMA) in PMNs isolated using different methods: method 1—DGC at 756 g for 30 min; method 2—sedimentation at 1g for 60 min (no centrifugation step); method 3—sedimentation at 1g for 60 min, overlay with plasma, followed by DGC at 756 g for 30 min; and method 4—sedimentation at 1g for 60 min, overlay with erythrocytes, followed by DGC at 756 g for 30 min.

Figure 6

Median fluorescence intensity [AFU] of antibodies against various surface antigens, including CD11b total (A), CD11b activated (B), CD62L (D), and CD66b (E), as well as the calculated ratio of CD11b activated to CD11b total (C), measured after different incubation times (0 h, 2 h, and 22 h) and using different isolation methods: method 1—DGC at 756 g for 30 min; method 2—sedimentation at 1g for 60 min (no centrifugation step); method 3—sedimentation at 1g for 60 min, overlay with plasma, followed by DGC at 756 g for 30 min; and method 4—sedimentation at 1g for 60 min, overlay with erythrocytes, followed by DGC at 756 g for 30 min.