title: [Establishment and evaluation of an in vitro method for neutrophil extracellular trap generation and degradation].
abstract: 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.
  
  
  CONCLUSIONS
  This work established an efficient method for in vitro generation and degradation of human NETs.
authors.affiliation: 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.
authors.forename: Jinlong
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Respiratory and Critical Care Medicine, Pingjin Hospital, Logistics University of Chinese People's Armed Police Forces, Tianjin 300162, China.
authors.forename: Yidan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Tianjin Key Laboratory of Cardiovascular Remodeling and Target Organ Injury, Institute of Cardiovascular Diseases and Heart Center, Tianjin 300162, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Tianjin Key Laboratory of Cardiovascular Remodeling and Target Organ Injury, Institute of Cardiovascular Diseases and Heart Center, Department of Respiratory and Critical Care Medicine, Pingjin Hospital, Logistics University of Chinese People's Armed Police Forces, Tianjin 300162, China.
authors.forename: Wenjie
authors.initials: W
authors.lastname: Ji
authors.affiliation: Tianjin Key Laboratory of Cardiovascular Remodeling and Target Organ Injury, Institute of Cardiovascular Diseases and Heart Center, Tianjin 300162, China.
authors.forename: Jihong
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Department of Respiratory and Critical Care Medicine, Pingjin Hospital, Logistics University of Chinese People's Armed Police Forces, Tianjin 300162, China.
authors.forename: Luqing
authors.initials: L
authors.lastname: Wei
authors.affiliation: Tianjin Key Laboratory of Cardiovascular Remodeling and Target Organ Injury, Institute of Cardiovascular Diseases and Heart Center, Tianjin 300162, China.
authors.forename: Yuming
authors.initials: Y
authors.lastname: Li
mesh_terms: D004247:DNA / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D065206:Extracellular Traps / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques / Q000379:methods; D018613:Microscopy, Confocal; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D006657:Histones; D004247:DNA; D003850:Deoxyribonuclease I; D013755:Tetradecanoylphorbol Acetate