Quantification of O‐GlcNAc protein modification in neutrophils by flow cytometry

Abstract

Observations of intracellular O-linked β-N-acetylglucosamine (O-GlcNAc) protein modification are primarily performed by Western blot or immunofluorescence microscopy. The goal of this study was to develop a flow cytometric-based assay for O-GlcNAc signaling and thus provide a more quantitative and rapid method to facilitate clinical analyses. Isolated peripheral blood neutrophils were stimulated with fMLF after adherence to glass cover slips. Cells in suspension were treated with either fMLF or PMA. Unstimulated cells served as controls. Neutrophils were fixed with formaldehyde and permeabilized with cold methanol before intracellular O-GlcNAc staining. Cells on cover slips were analyzed by fluorescence microscopy, and suspension cell data were acquired by flow cytometry. O-GlcNAc protein modification was increased following neutrophil stimulation with either 100 nM fMLF or 10 nM PMA. Increases were detected following either treatment using both flow cytometry and fluorescence microscopy. The time necessary for the completion of staining, data acquisition, and analysis was considerably less using flow cytometry. In addition, flow cytometry allows for the analysis of a substantially greater number of cells. Neutrophil protein modifications by O-GlcNAc are rapidly detected using flow cytometry and provide information similar to that observed using fluorescence microscopy.

O-GlcNAcylation and its role in the immune system

“…Neutrophils not only phagocytose pathogens and produce pro-inflammatory cytokines, ROS and granular proteins, but also release neutrophil extracellular traps (NET) to minimize the damage caused by pathogens [112]. Previous studies demonstrated that O-GlcNAcylated proteins in neutrophils are rapidly increased within 2 min following treatment with N-Formylmethionine-leucyl-phenylalanine (fMLF), a polymorphonuclear and mononuclear phagocyte activator [113][114][115], suggesting that O-GlcNAcylation may be crucial for the function of neutrophils. Increased O-GlcNAcylation promotes chemotaxis and cellular mobility of neutrophils [114,116].…”

O-GlcNAcylation in immunity and inflammation: An intricate system (Review)

“…It has been reported that O-GlcNAcylation mediates the activation of the small GTPase Rac and the downstream p38 and p44/42 mitogen-activated protein kinase (MAPK) signaling pathways in neutrophils. These MAPKs are known to modulate cellular chemotaxis leading to cell movement and inflammatory response (66-68).…”

The role of O‐GlcNAcylation in immunity against infections

“…29 A more extensive analysis of overall O-GlcNAcylation levels in human neutrophils by flow cytometry and fluorescence microscopy also showed increased O-GlcNAcylation following fMLP stimulation when compared with unstimulated controls. 46 Although these results indicate that neutrophil chemotaxis is positively dependent on O-GlcNAcylation, there also is a study which found an inhibitory effect of GlcN treatment on human neutrophil chemotaxis and fMLP-driven F-actin interactions. 47 This discrepancy could be explained by the 10-fold lower concentration of GlcN used in this study, 47 when compared with the aforementioned studies.…”