Metabolic variations in human neutrophils with different combinations of PMA treatments (2DG, 6AN, DPI and AA): Part 2

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..., , # Metabolic variations in human neutrophils with different combinations of PMA treatments (2DG, 6AN, DPI and AA): Part 2

[https://doi.org/10.5061/dryad.z34tmpgpk](https://doi.org/10.5061/dryad.z34tmpgpk)

## Description of the data and file structure

Metabolomic variations in human neutrophils in different conditions: PMA (100nM) treatment and PMA together with 100mM 2DG (PMA+2DG), 5mM 6AN (PMA+6AN), 10μM DPI (PMA+DPI), 1μM AA (PMA+AA). Metabolites were extracted at 15 minutes.

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
* PMA_15m.mzXML: human neutrophils treated with PMA for 15 minutes.
* PMA_2DG_15m.mzXML: human neutrophils treated with PMA and 2DG for 15 minutes.
* PMA_6AN_15m.mzXML: human neutrophils treated with PMA and 6AN for 15 minutes.
* PMA_AA_15m.mzXML: human neutrophils treated with PMA and AA for 15 minutes.
* PMA_DPI_15m.mzX...,