Table 1.

The benefits and the limitations of each laboratory method used in the context of NET evaluation.

Techniques	Benefits	Limitations	References
Enzyme-linked immunosorbent assay (ELISA)	Simplicity, specificity, cost-effectiveness, objective and quantitative	Results standardization and differences between in vivo and in vitro NETs	Kasprzycka et al., 2019 [41]; Thålin et al., 2017 [45]; Masuda et al., 2017 [49]
Immunofluorescence microscopy (IFM)	Distinguishes cell death mechanisms, objective quantification of NETs with software-based methods, and detects NETs in vitro, ex vivo and in situ	A time-consuming procedure, laborious, limited reproducibility, not available for rapid screening of many cells or samples, observer-dependent, and extensive precaution with sample preparation	Brinkmann et al., 2004 [11]; Von Köckritz-Blickwede et al., 2010 [50]; Brinkmann et al., 2012 [51]; Coelho et al., 2015 [52]; De Buhr and von Köckritz-Blickwede, 2016 [15]; Lv et al., 2020 [53]
Electron microscopy	Distinguishes cell death mechanisms and detects NETs in vitro and in situ	Biased based on the field of view and no reliable distinguishing between NETs and fibrin by SEM	Fuchs et al., 2007 [24]; De Buhr and von Köckritz-Blickwede, 2016 [15]; Krautgartner et al., 2008 [54]; Lv et al., 2020 [53]
Live imaging	Live in vivo imaging using animal models, close examination of NET structures, and distinguishes NETs from other cell deaths	Staining of many components on NETs, complex equipment and expensive	Cho et al., 2011 [55]; Kolaczkowska et al., 2015 [56]; Gupta et al., 2018 [57];
Flow cytometry	Qualitative, quantitative and objective; detects pre-NETotic cells, both in vivo and in vitro, rapid and sort between cell populations and has the possibility of direct applications in blood samples	Extensive precaution with sample preparation; detects only pre-NETotic cells and is incapable of detecting released NETs/remnants	Masuda et al., 2017 [49]; Kasprzycka et al., 2019 [41]; Gavillet et al., 2015 [58]; Zharkova et al., 2019 [59]
Multispectral image flow cytometer (MIFC)	Rapid, semiautomated and studies subcellular morphology and distinguishes cell death mechanisms	Sophisticated equipment and detects fewer NETotic cells than existing	Zhao et al., 2015 [60]
Western blot	Semi-quantitative, specificity and sensitivity	Multi-step protocol and optimizes the concentration of the primary antibody	Liu et al., 2016 [61]; Lv et al., 2020 [53]