Abstract
Neutrophil extracellular traps (NETs) are fibrous mesh-like, web-like, or string-like structures which are composed of DNA, histones, and granule proteins such as neutrophil elastase or myeloperoxidase. When activated by phorbol myristate acetate, interleukin-8, lipopolysaccharide (LPS), and various pathogens, neutrophils release NETs. We reported that NETs were classified as two distinct forms; cell-free NETs that were released away from neutrophils and anchored NETs that were anchored to neutrophils. In general, extracellular DNAs are used as a surrogate marker of NETs. Here, we describe a protocol regarding quantitative procedures of extracellular DNAs released from ex vivo neutrophils activated by LPS using fluorometric double-stranded DNA (dsDNA) quantification assay.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. The representative data of ex vivo NETs released from human neutrophils activated by LPS. (A). The DNA concentration at the indicated dose of LPS was shown as the sum of the amount of anchored and cell-free NETs. (B). The DNA concentration of anchored NETs was shown at the indicated dose of LPS on the first plate. Anchored NETs were anchored to neutrophils adhering to the plate. (C). The DNA concentration of cell-free NETs was shown at the indicated dose of LPS. They were transferred to the second plate as the supernatant of the first plate.
Notes
Gentle aspiration of the supernatant should be needed to avoid the contamination of anchored NETs into the second plate.
Acknowledgments
This protocol was adapted from the previously reported in Tanaka et al. (2014). This work was partly supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (KAKENHI 25462052 to K.T.).
References
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