Pehl Sophie, Hundhammer Tobias, Rimboeck Julia, Kraus Richard, Heckscher Simon, Kellermeier Fabian, Gruber Michael, Wittmann Sigrid
Department of Anesthesiology, University Hospital Regensburg, 93042 Regensburg, Germany.
Institute of Functional Genomics, University of Regensburg, 93042 Regensburg, Germany.
Cells. 2025 Aug 30;14(17):1350. doi: 10.3390/cells14171350.
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.
为了在实验室条件下研究粒细胞，通常需要进行离心步骤以从全血中分离中性粒细胞。然而，迄今为止只有少数研究探讨了离心本身对中性粒细胞功能状态的直接影响。本研究旨在阐明离心过程中导致粒细胞改变的机制。我们假设颗粒在离心过程中会发生形态改变，从而导致高效抗菌酶释放到细胞胞质溶胶中。使用有无离心的不同方法从全血中分离中性粒细胞，并通过流式细胞术、酶联免疫吸附测定和质谱分析。我们的研究结果表明，细胞内颗粒在离心过程中会受到损伤，导致胞质溶胶中存在颗粒内酶。此外，可以证实离心会导致高反应性次氯酸（HOCl）的形成。细胞内HOCl的产生可能解释了基于离心分离后在中性粒细胞中观察到的许多改变，包括表面抗原表达的改变和对刺激反应的改变。在未来的研究中，应避免细胞分离过程中的离心步骤。更耗时但更温和的沉降方法更可取，只要不需要获得高度纯化的中性粒细胞部分，就可以使用。
