Neutrophils are the most abundant circulating white blood cells and are central players of the innate immune response. During their lifecycle, neutrophils mainly evolve under low oxygen conditions (0.1 to 4% O2) to which they are well adapted. Neutrophils are atypical cells since they are mainly glycolytic, and highly susceptible to oxygen-exposure, which induces their activation and death, through mechanisms which remain currently elusive. Nevertheless, nearly all studies conducted on neutrophils are carried out under atmospheric oxygen (21%), corresponding to hyperoxic conditions. Here we investigated the impact of hyperoxia during neutrophil purification and culture on neutrophil viability, activation and cytosolic protein content. Neutrophil hyperactivation (CD62L shedding) is induced during culture under hyperoxic conditions (24h), compared to neutrophils cultured under anoxic conditions. In addition, we show that maintaining neutrophils in autologous plasma is the most suitable strategy to maintain their basal state. Our results show that manipulating neutrophils under hyperoxic conditions leads to the loss of 100 cytosolic proteins during purification, while it does not lead to an immediate impact on neutrophils activation (CD11bhigh, CD54high, CD62Llow) or viability (DAPI+). We identified two clusters of proteins belonging to the cholesterol metabolism and to the complement and coagulation cascade pathways, which are highly susceptible to neutrophil oxygen-exposure during their purification. In conclusion, preserving neutrophil from oxygen-exposure during their manipulation, purification and culture is recommended to avoid their experimental activation and for preserving a large set of cytosolic proteins from alteration.

中文翻译：

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防止中性粒细胞不接触氧气可以维持其基础状态

中性粒细胞是循环中最丰富的白细胞，并且是先天免疫反应的核心参与者。在它们的生命周期中，嗜中性粒细胞主要是在低氧条件下（0.1至4％O2）适应性发展的。中性粒细胞是非典型细胞，因为它们主要是糖酵解的，并且高度易受氧暴露，通过目前尚不清楚的机制，氧诱导其活化和死亡。但是，几乎所有对中性粒细胞的研究都是在大气氧（21％）下进行的，这与高氧条件相对应。在这里，我们研究了中性粒细胞纯化和培养过程中高氧对中性粒细胞活力，活化和胞浆蛋白含量的影响。在高氧条件下（24h）培养期间会诱导中性粒细胞过度活化（CD62L脱落），与缺氧条件下培养的嗜中性粒细胞相比。另外，我们表明在自体血浆中维持嗜中性粒细胞是维持其基础状态的最合适策略。我们的结果表明，在高氧条件下操作嗜中性粒细胞会导致纯化过程中损失100种胞质蛋白，而不会立即影响嗜中性粒细胞的活化（CD11bhigh，CD54high，CD62Llow）或生存力（DAPI +）。我们鉴定了属于胆固醇代谢以及补体和凝血级联途径的两个蛋白质簇，它们在纯化过程中极易暴露于嗜中性粒细胞。总之，在操作过程中避免嗜中性粒细胞接触氧气，