How Neutrophil Extracellular Traps Become Visible

Table 1

Summary of the main NET visualization techniques used for quantification of NETs and its advantages or disadvantages.

Dye	Technique	Parameter	Advantages	Disadvantages	Selected references
SYTOX dye/PicoGreen	FM, eye	Percentage of NET formation	Visible differentiation between necrosis and NETosis	Occasionally biased by selection of field of view, staining of DNA in NETs by DNA-intercalating dye can be blocked by cationic peptides	[3, 40, 44]
Antibody against histone-DNA complexes + Dapi	IFM, eye	Percentage of NET formation	Visible differentiation between necrosis and NETosis	Occasionally biased by selection of field of view	[7, 8, 10, 34, 40, 44]
Antibody against elastase and histone-DNA complexes + Hoechst 33342	IFM, Image J	Percentage of NET formation	Unbiased software-based quantification	Clump of NETs derived from multiple cells count as one single event, occasionally biased by selection of field of view	[67]
Antibody against histone-DNA complexes + Dapi	IFM, Image J	Level of NET degradation	Unbiased software-based quantification	Occasionally biased by selection of field of view	[24, 25]
Antibody against histone-DNA complexes + Dapi	IFM, open source software	Level of NET degradation	Unbiased software-based quantification	Occasionally biased by selection of field of view	[68]
SYTOX dye/PicoGreen	FR	DNA release (g/mL)	Unbiased	No differentiation between necrosis and NETosis, staining of DNA in NETs by DNA-intercalating dye can be blocked by cationic peptides	[27, 37, 40]
PicoGreen after nuclease digestion	FR	DNA release (g/mL)	Unbiased	Staining of DNA in NETs by DNA-intercalating dye can be blocked by cationic peptides, less sensitive compared to antibody-mediated detection of NETs	[34, 40]
Antibody against MPO + Hoechst	Imaging flow cytometry	Percentage of NET formation	Unbiased, automated, enables differentiation between suicidal NETosis and vital NETosis	Imaging of cells currently undergoing NETosis and thus this method may miss those that have already lysed	[49]
Antibody against H3cit + MPO	Flow cytometry	Percentage of NET formation	Unbiased, automated, can be combined with sorting	Does not detect H3cit-independent events	[45]
Uranyl-acetate, osmium tetroxide, ruthenium red-osmium tetroxide, Cuprolinic Blue	TEM	Morphology of NET-releasing cells	Visible differentiation between necrosis and NETosis, can be used in combination with immunostaining of certain structures in NETs	Occasionally biased by selection of field of view	[40, 50]
Osmium tetroxide/gold	SEM	Amount and structure of NETs-releasing cells	Visible differentiation between necrosis and NETosis, can be used in combination with immunostaining of certain structures in NETs	Occasionally biased by selection of field of view, fibrin mimics NET structures	[40, 50]

IFM: immunofluorescence microscopy, FM: fluorescence microscopy, FR: fluorescence reader, MPO: myeloperoxidase, TEM: transmission electron microscopy, SEM: scanning electron microscopy, and H3cit: histone citrullination.