title: Microfluidic device for simultaneous analysis of neutrophil extracellular traps and production of reactive oxygen species.
abstract: Neutrophil extracellular traps (NETs) were first reported in 2004, and since their discovery, there has been an increasing interest in NETs, how they are formed, their role in controlling infections, and their contribution to disease pathogenesis. Despite this rapid expansion of our understanding of NETs, many details remain unclear including the role of reactive oxygen species (ROS) in the formation of NETs. Further, to study NETs, investigators typically require a large number of cells purified via a lengthy purification regimen. Here, we report a microfluidic device used to quantify both ROS and NET production over time in response to various stimulants, including live bacteria. This device enables ROS and NET analysis using a process that purifies primary human neutrophils in less than 10 minutes and requires only a few microliters of whole blood. Using this device we demonstrate the ability to identify distinct capabilities of neutrophil subsets (including ROS production and NET formation), the ability to use different stimulants/inhibitors, and the ability to effectively use samples stored for up to 8 hours. This device permits the study of ROS and NETs in a user-friendly format and has potential for widespread applications in the study of human disease.
authors.affiliation: Medical Scientist Training Program, University of Wisconsin, Madison, WI, USA.
authors.forename: S F
authors.initials: SF
authors.lastname: Moussavi-Harami
authors.forename: K M
authors.initials: KM
authors.lastname: Mladinich
authors.forename: E K
authors.initials: EK
authors.lastname: Sackmann
authors.forename: M A
authors.initials: MA
authors.lastname: Shelef
authors.forename: T W
authors.initials: TW
authors.lastname: Starnes
authors.forename: D J
authors.initials: DJ
authors.lastname: Guckenberger
authors.forename: A
authors.initials: A
authors.lastname: Huttenlocher
authors.forename: D J
authors.initials: DJ
authors.lastname: Beebe
mesh_terms: D001562:Benzimidazoles / Q000737:chemistry; D002843:Chromatin / Q000378:metabolism; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D004867:Equipment Design; D065206:Extracellular Traps; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D056656:Lab-On-A-Chip Devices; D044085:Microfluidics / Q000379:methods; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism / Q000382:microbiology; D009861:Onium Compounds / Q000737:chemistry; D011550:Pseudomonas aeruginosa / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D001562:Benzimidazoles; D002843:Chromatin; D004129:Dimethylpolysiloxanes; D005456:Fluorescent Dyes; D009861:Onium Compounds; D017382:Reactive Oxygen Species; C013830:baysilon; C007517:diphenyleneiodonium; D019255:NADPH Oxidases; C086986:CYBA protein, human; C017807:bisbenzimide ethoxide trihydrochloride