2019
DOI: 10.3791/59150
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A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

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Cited by 8 publications
(7 citation statements)
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“…Fresh human neutrophils were used to assess neutrophil extracellular trap (NET) formation by use of a previously described protocol based on the PKH26 kit (Sigma-Aldrich) with minor modifications ( 83 ). In brief, 37,500 neutrophils in RPMI medium were stained with a 2 μM concentration of the red fluorescent cell linker provided in the PKH26 kit before they were challenged with A. actinomycetemcomitans for 2 h. Subsequently, the infected neutrophils were stained with a 5 μM concentration of the membrane-impermeable DNA dye Sytox green (Thermo Fisher Scientific), and the samples were fixed with PFA.…”
Section: Methodsmentioning
confidence: 99%
“…Fresh human neutrophils were used to assess neutrophil extracellular trap (NET) formation by use of a previously described protocol based on the PKH26 kit (Sigma-Aldrich) with minor modifications ( 83 ). In brief, 37,500 neutrophils in RPMI medium were stained with a 2 μM concentration of the red fluorescent cell linker provided in the PKH26 kit before they were challenged with A. actinomycetemcomitans for 2 h. Subsequently, the infected neutrophils were stained with a 5 μM concentration of the membrane-impermeable DNA dye Sytox green (Thermo Fisher Scientific), and the samples were fixed with PFA.…”
Section: Methodsmentioning
confidence: 99%
“…After 3¾ h of stimulation, 1 µM SYTOXgreen (ThermoFisher, USA) was added for 15 min, after which neutrophils were fixed with 4% paraformaldehyde (AddedPharma, Netherlands). Hereafter, the NETs were visualized and quantified by 3 D confocal microscopy using the automated BD Pathway 855 (BD Biosciences, USA), or the Image Xpress Micro Confocal (Molecular Devices, USA), as described previously [ 30 , 31 ].…”
Section: Methodsmentioning
confidence: 99%
“…NET formation was quantified using an approach previously described by our group . Briefly, PKH‐labeled neutrophils were plated in a 96‐well culture plate (Falcon) at 37,500 cells per well in phenol red–free RPMI 1640 medium (Life Technologies) supplemented with 2% heat‐inactivated FCS.…”
Section: Methodsmentioning
confidence: 99%