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, while metabolic and functional changes were confirmed with mass spectrometry, oxidative burst, and NETosis measurements. Our findings show that neutrophils undergo rapid metabolic remodeling to a reduced redox state, followed by a shift to an oxidized redox state during activation. Additionally, single cell analysis reveals a heterogeneous metabolic response across neutrophils and donors to live pathogen infection (Pseudomonas aeruginosa and Toxoplasma gondii). Finally, consistent metabolic changes were validated with neutrophils in vivo using zebrafish larvae. This study demonstrates the potential of OMI as a versatile tool for studying neutrophil metabolism and underscores its use across different biological systems, offering insights into neutrophil metabolic activity and function at a single cell level. This data set is divided in two parts. Part 1: Time course of metabolic variations in human neutrophils with PMA treatment alone or in combination with DPI. Part 2: Metabolic variations in human neutrophils with different combinations of PMA treatments (2DG, 6AN, DPI, and AA)

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 min–1. Data acquisition was performed using full scan mode. Metabolite identification relied on exact m/z and retention time, which were determined using chemical standards. Data acquisition was conducted using Xcalibur 4.0 software and analysis on Maven.