Time course of metabolic variations in human neutrophils with PMA treatment alone or in combination with DPI: Part 1

Neutrophils, the most abundant leukocytes in human peripheral circulation, are crucial for the innate immune response. They are typically quiescent but rapidly activate in response to infection and inflammation, performing diverse functions such as oxidative burst, phagocytosis, and NETosis, which require significant metabolic adaptation. Deeper insights into such metabolic changes will help identify regulation of neutrophil functions in health and diseases. Due to their short lifespan and associated technical challenges, the metabolic processes of neutrophils are not completely understood. This study uses optical metabolic imaging (OMI), which entails optical redox ratio and fluorescence lifetime imaging microscopy of intrinsic metabolic coenzymes NAD(P)H and FAD to assess the metabolic state of single neutrophils. Primary human neutrophils were imaged in vitro under a variety of activation conditions and metabolic pathway inhibitors, w..., For extracting intracellular metabolites, neutrophils were washed with PBS after removing the culture medium. Pelleted neutrophils was extracted using 150 μL of cold acetonitrile/methanol/water (40:40:20 v:v:v) of liquid chromatography–mass spectrometry (LC– MS) grade (for each 2 million cells), followed by centrifugation at 20,627g for 5 minutes at 4°C to eliminate any insoluble residue. Samples were dried under N2 flow followed by resuspension in LC–MS-grade water as loading solvent. The soluble metabolites obtained were analyzed using a Thermo Q-Exactive mass spectrometer connected to a Vanquish Horizon Ultra-High Performance Liquid Chromatograph. Metabolites were separated on a 2.1 × 100mm, 1.7 μM Acquity UPLC BEH C18 Column (Waters) employing a gradient of solvent A (97:3 H2O/methanol, 10 mM TBA, 9 mM acetate, pH 8.2) and solvent B (100% methanol). The gradient used was: 0 min, 5% B; 2.5 min, 5% B; 17 min, 95% B; 21 min, 95% B; 21.5 min, 5% B. The flow rate was maintained at 0.2 ml..., , # Time course of metabolic variations in human neutrophils with PMA treatment alone or in combination with DPI: Part 1 [https://doi.org/10.5061/dryad.q83bk3jrq](https://doi.org/10.5061/dryad.q83bk3jrq) ## Description of the data and file structure Metabolomic variations in human neutrophils in different conditions: unstimulated control, PMA (100nM) treatment and PMA together with NOX2 inhibitor DPI (10μM) treatment (PMS+DPI). We also also includes stimulation with LPS (20μg/L) and TNFα (5μg/L). Metabolites were extracted at 15, 30 and 60 mins for all the conditions except for LPS and TNFα, which were extracted at 60 mins. Samples were run in duplicated (indicated by A or B in the name of the sample). Timepoints and conditions are indicated in the name of the sample.  * Blank1.mzXML : Blank sample Replicate 1 * Blank2.mzXML: Blank sample Replicate 2 * Blank3.mzXML : Blank sample Replicate 3 * LPS_60m_A.mzXML: human neutrophils stimulated with LPS for 60 minutes replicate 1 * LPS_...,中性粒细胞(Neutrophils)是人体外周循环中丰度最高的白细胞(leukocytes)，在固有免疫应答(innate immune response)中发挥关键作用。它们通常处于静息状态，但会在感染与炎症刺激下快速活化，执行氧化爆发(oxidative burst)、吞噬作用(phagocytosis)及中性粒细胞胞外陷阱形成(NETosis)等多种功能，这些过程均需要显著的代谢适应(metabolic adaptation)。对这类代谢变化的深入解析，将有助于阐明健康与疾病状态下中性粒细胞功能的调控机制。由于中性粒细胞寿命较短且存在相关技术难题，目前其代谢过程尚未被完全阐明。本研究采用光学代谢成像(OMI)技术，通过对内源性代谢辅酶(intrinsic metabolic coenzymes)烟酰胺腺嘌呤二核苷酸磷酸/烟酰胺腺嘌呤二核苷酸(NAD(P)H)和黄素腺嘌呤二核苷酸(FAD)进行光学氧化还原比率(optical redox ratio)分析与荧光寿命成像显微镜(fluorescence lifetime imaging microscopy)检测，以评估单个中性粒细胞的代谢状态。原代人中性粒细胞在体外多种活化条件及代谢通路抑制剂处理下完成成像，[原文此处有截断]。对于胞内代谢物的提取：移除培养基后，中性粒细胞用磷酸盐缓冲液(PBS)洗涤。每2×10^6个细胞使用150微升色谱-质谱联用(LC-MS)级别的预冷乙腈/甲醇/水(40:40:20，体积比)对沉淀的中性粒细胞进行代谢物提取，随后在4℃下以20627g离心5分钟以去除不溶性残渣。样品经氮气(N2)吹干后，用LC-MS级水作为复溶溶剂进行重悬。所得可溶性代谢物采用搭载Vanquish Horizon超高效液相色谱仪(Ultra-High Performance Liquid Chromatograph)的Thermo Q-Exactive质谱仪进行分析。代谢物在2.1×100mm、1.7μm的Acquity UPLC BEH C18色谱柱(Waters公司)上进行分离，流动相A为97:3的水/甲醇溶液（含10mM四丁基铵(TBA)、9mM乙酸盐，pH 8.2），流动相B为100%甲醇。洗脱梯度设置如下：0分钟时5% B；2.5分钟时5% B；17分钟时95% B；21分钟时95% B；21.5分钟时5% B。流速维持在0.2毫升/分钟[原文此处有截断]。 # 单独或联合使用DPI处理的PMA刺激下人中性粒细胞代谢变化的时间进程：第一部分 https://doi.org/10.5061/dryad.q83bk3jrq ## 数据与文件结构说明 本数据集涵盖不同处理条件下人中性粒细胞的代谢组变化：未刺激对照组、佛波酯(PMA, 100nM)处理组、以及PMA联合NOX2抑制剂二苯基碘鎓(DPI, 10μM)处理组（原文标注为PMS+DPI）。此外还包含脂多糖(LPS, 20μg/L)与肿瘤坏死因子α(TNFα, 5μg/L)刺激组。除LPS与TNFα刺激组仅在60分钟时点提取代谢物外，其余各组均在15、30及60分钟三个时间点提取代谢物。所有样品均设置生物学重复（样品名称中以A或B标识重复组别）。样品名称中包含对应的处理条件与采样时间点信息。 * Blank1.mzXML：空白样品重复1 * Blank2.mzXML：空白样品重复2 * Blank3.mzXML：空白样品重复3 * LPS_60m_A.mzXML：经LPS刺激60分钟的中性粒细胞样品重复1 * LPS_...[原文此处有截断]