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Updated: Apr 24, 2026
A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation
Published on: January 29, 2019
[Establishment and evaluation of an in vitro method for neutrophil extracellular trap generation and degradation]
Jinlong Li1, Yidan Zhang2, Xin Zhou3
1Graduate School, Tianjin Medical University, Tianjin 300070; Tianjin Key Laboratory of Cardiovascular Remodeling and Target Organ Injury, Institute of Cardiovascular Diseases and Heart Center, Tianjin 300162, China.
Objective:
To evaluate a novel method for in vitro generation and degradation of neutrophil extracellular traps (NETs), which are a newly recognized structure that is involved in the pathogenesis of autoimmune diseases and thrombosis.
Methods:
Neutrophils from peripheral blood of healthy donors were obtained by Ficoll-Histopaque gradient separation. NET release was initiated by phorbol myristate acetate (PMA) and validated by immunofluorescence staining and agarose gel electrophoresis. NETs degraded by DNase I and healthy human plasma were quantified by fluorescence spectrometry after staining with PicoGreen.
Results:
HE staining showed that the purity of neutrophils was up to 95% after Ficoll-Histopaque gradient separation. NET immunofluorescent staining revealed that the network structure was mainly composed of DNA and histones, with molecular length more than 10 kb as demonstrated by agarose gel electrophoresis. Moreover, both DNase and healthy human plasma could induce the degradation of NETs, in varying degrees.
Conclusion:
This work established an efficient method for in vitro generation and degradation of human NETs.
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