Neutrophils are the first immune responders to bacterial or viral infection and play key roles in the host immune response; however, handling and investigating fresh neutrophils can be challenging. Here, we present a protocol for isolating neutrophils from the peripheral blood of healthy donors using density gradient separation method. We describe steps for morphology analysis by cytospin and immunophenotyping by flow cytometry analysis. This protocol can be used for the isolation of neutrophils from healthy and diseased individuals. For complete details on the use and execution of this protocol, please refer to Parthasarathy et al.1.
Copyright © 2023 Merck Sharp & Dohme LLC., a subsidiary of Merck & Co., Inc., Rahway, NJ, USA. Published by Elsevier Inc. All rights reserved.

Sepsis is a life-threatening condition caused by a dysregulated host response to infection. Despite continued efforts to understand the pathophysiology of sepsis, no effective therapies are currently available. While singular components of the aberrant immune response have been investigated, comprehensive studies linking different data layers are lacking. Using an integrated systems immunology approach, we evaluated neutrophil phenotypes and concomitant changes in cytokines and metabolites in patients with sepsis. Our findings identify differentially expressed mature and immature neutrophil subsets in patients with sepsis. These subsets correlate with various proteins, metabolites, and lipids, including pentraxin-3, angiopoietin-2, and lysophosphatidylcholines, in patients with sepsis. These results enabled the construction of a statistical model based on weighted multi-omics linear regression analysis for sepsis biomarker identification. These findings could help inform early patient stratification and treatment options, and facilitate further mechanistic studies targeting the trifecta of surface marker expression, cytokines, and metabolites.
© 2023 Merck Sharp & Dohme LLC., a subsidiary Merck & Co, The Author(s).