title: A novel image-based quantitative method for the characterization of NETosis.
abstract: NETosis is a newly recognized mechanism of programmed neutrophil death. It is characterized by a stepwise progression of chromatin decondensation, membrane rupture, and release of bactericidal DNA-based structures called neutrophil extracellular traps (NETs). Conventional 'suicidal' NETosis has been described in pathogenic models of systemic autoimmune disorders. Recent in vivo studies suggest that a process of 'vital' NETosis also exists, in which chromatin is condensed and membrane integrity is preserved. Techniques to assess 'suicidal' or 'vital' NET formation in a specific, quantitative, rapid and semiautomated way have been lacking, hindering the characterization of this process. Here we have developed a new method to simultaneously assess both 'suicidal' and 'vital' NETosis, using high-speed multi-spectral imaging coupled to morphometric image analysis, to quantify spontaneous NET formation observed ex-vivo or stimulus-induced NET formation triggered in vitro. The use of imaging flow cytometry allows automated, quantitative and rapid analysis of subcellular morphology and texture, and introduces the potential for further investigation using NETosis as a biomarker in pre-clinical and clinical studies.
authors.affiliation: Systemic Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, United States.
authors.forename: Wenpu
authors.initials: W
authors.lastname: Zhao
authors.affiliation: EMD Millipore Corporation, Billerica, MA 01821, United States.
authors.forename: Darin K
authors.initials: DK
authors.lastname: Fogg
authors.affiliation: Systemic Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, United States. Electronic address: mariana.kaplan@nih.gov.
authors.forename: Mariana J
authors.initials: MJ
authors.lastname: Kaplan
mesh_terms: D065206:Extracellular Traps / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D019044:Image Cytometry / Q000379:methods; D008027:Light; D009504:Neutrophils / Q000502:physiology*; D061848:Optical Imaging / Q000379:methods
keywords: Cell death; Flow cytometry; Microscopy; Neutrophil extracellular traps