Table 1.
Summary of the main NET visualization techniques used for quantification of NETs and their 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 | (1, 31, 32) |
| 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 | (31–36) |
| 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 | (37) |
| 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 | (38, 39) |
| 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 | (22) |
| SYTOX dye/PicoGreen | FR | DNA release (μg/mL) | Unbiased | No differentiation between necrosis and NETosis, staining of DNA in NETs by DNA-intercalating dye can be blocked by cationic peptides | (31, 40, 41) |
| PicoGreen after nuclease digestion | FR | DNA release (μg/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 | (31, 36) |
| Antibody against MPO + Hoechst | MIFC | 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 | (21) |
| Antibody against H3cit + MPO | Flow cytometry | Percentage of NET formation | Unbiased, automated, can be combined with sorting | Does not detect H3cit-independent events | (42) |
| 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 | (31, 43, 44) |
| 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 | (31, 43, 44) |
Adopted from de Buhr and Köckritz-Blickwede (30). IFM, immunofluorescence microscopy; FM, fluorescence microscopy; FR, fluorescence reader; MIFC, microscopy imaging flow cytometry; MPO, myeloperoxidase; TEM, transmission electron microscopy; SEM, scanning electron microscopy; H3cit, histone citrullination.