• Jinlong Li, Yidan Zhang, Xin Zhou, Wenjie Ji, Jihong Zhao, Luqing Wei, and Yuming Li.
• Graduate 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.
• Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014 Sep 1;30(9):986-8.

ObjectiveTo 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.MethodsNeutrophils 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.ResultsHE 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.ConclusionThis work established an efficient method for in vitro generation and degradation of human NETs.

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