Background/Purpose:

Neutrophil extracellular traps (NETs) consist of decondensed chromatin and antimicrobial peptides and are able to trap and kill pathogens. Several studies suggest a pathogenic role for NETosis in autoimmune disease (AID), such as systemic lupus erythematosus (SLE), ANCA-associated vasculitis (AAV) and rheumatoid arthritis (RA). For investigation of NET triggers in relation to AID, NET release should be quantified with a standard and reliable method. Thus far, methods for NET quantification are heterogeneous, e.g. based on fluorescence microscopy, measuring DNA release or measuring other NET components. The aim was to develop an assay for inducing and quantifying NETs, preferably applicable to use in a high-throughput system.

Methods:

Neutrophils were isolated from healthy volunteers and were labelled with a membrane staining, PKH26, for visualization in immunocytochemistry (ICC). Neutrophils were seeded in a 96-well plate and incubated with different stimuli. IgG aggregates were used as a positive control. Extracellular DNA was stained with Sytox green. Further stainings used were Hoechst, anti-myeloperoxidase (MPO), anti-neutrophil elastase (NE) and anti-citrullinated histon3 (acitH3). Images were obtained with a BD Pathway 855 that was programmed to automatically and randomly obtain pictures from different locations within the well. Titration experiments with IgG aggregates were performed to induce NET release and image NETs with a 10x, 20x and 40x magnification lens. Subsequently NETs were quantified and the results obtained with different lenses were compared to find the optimal magnification for NET visualization and quantification.

Results:

We found that IgG aggregates were able to induce NET release in a dose-dependent manner. NET release in this assay was confirmed with acitH3, NE and MPO stainings. We quantified the amount of NETs with ImageJ software, by determining the ratio between positive Sytox green area and the positive PKH26 area, of which we found that it accurately represents the amount of cells present. NET quantification using different magnification lenses showed that the 20x magnification was able to capture the same amount of information compared to the 40x magnification and was able to increase the area for analysis up to 11% of the total area compared to the standard 2%. 

Conclusion:

We developed a semi-automatic and reproducible system that reliably quantifies the NET-inducing capacity of many samples simultaneously and that is applicable as a high throughput system.

« Back to 2015 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-high-throughput-system-for-quantification-of-in-vitro-neutrophil-extracellular-trap-formation-with-fluorescence-immunocytochemistry/