Neutrophil

Inflammation Assays

Respiratory Burst (ROS, Phagoburst)
NETs
Phagocytosis/trogocytosis

Therapeutic blocking of NETosis and enhancement of neutrophil phagocytosis/trogocytosis

Neutrophils are armed with a variety of effector mechanisms, they release neutrophil extracellular traps (NETs), comprising of chromatin and antimicrobial proteins which are released via a unique, pro-inflammatory form of cell death called NETosis. Dysregulated NET release can damage the host, contributing to autoimmune diseases such as Systemic Lupus Erythematosus (SLE) by the release of autoantigens. Similarly, dysregulated NET release contributes to diseases such as atherosclerosis, deep vein thrombosis and has been shown to promote cancer progression and metastasis. Modulating neutrophil function is therefore a potential therapeutic intervention.

Immune complex-driven NETosis in autoimmune inflammation

Neutrophils were isolated from fresh blood, cultured with or without NOX2 inhibitor (DPI) or Fc block, and then stimulated with immune complexes (IC) in the presence of SYTOX green to detect NETs. Cells were imaged for green fluorescence (NETs) every 15 minutes for 4 hours. Data shows that NETosis increases in response to stimulation with IC and is inhibited by Fc blockade or DPI.

Phagocytosis - Neutrophils were cultured with pHrodo labelled *E. coli* in the presence or absence of a titration of cytochalasin D and phagocytosis measured by flow cytometry. Phagocytic neutrophils were identified as CD66b+ pHrodo+.

Inhibiting neutrophil transmigration through endothelial monolayer

Endothelial cells (HUVEC) were cultured on transwell inserts and confluency confirmed. Neutrophils were isolated from fresh blood and plated on top of the endothelial layer in the presence or absence of CXCR1/2 antagonist (Sch527123), Cytochalasin D, or controls. Chemoattractant IL-8 was added to the lower chamber of the transwell and cells were allowed to migrate before being analysed by luminescence. Sch527123 demonstrated a dose responsive inhibition of neutrophil migration across the endothelial monolayer. Neutrophil migration was inhibited by the positive control, Cytochalasin D.

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