Journal: The FASEB Journal
Article Title: Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines and protection from antiproteases
Figure Lengend Snippet: Aggregation of neutrophils stimulated with triggers of NET formation. A ) Representative photographs from high-density (1 ÃÂ 10 8 cells/ml) cultures of human peripheral blood neutrophils incubated with MSU crystals, PMA, Iono, or without stimulants for 3 h. Scale bar, 3 mm. B ) Scanning electron microscope micrographs from high-density (1 ÃÂ 10 8 cells/ml) neutrophil cultures stimulated as indicated. Scale bars, 20 ÃÂµm. C ) Representative multiphoton images of neutrophils stained with DNA dye SiR-DNA and membrane marker PKH67 and cultured in densities of 1 ÃÂ 10 8 cells/ml with MSU crystals or without stimulant. Scale bars, 50 ÃÂµm. D ) Assessment of MSU crystalÃ¢ÂÂinduced aggNETs with combination of multiphoton imaging and polarization microscopy. Scale bar, 50 ÃÂµm. All experiments were performed independently 3 times.
Article Snippet: Isolated human PMNs from healthy donors were cultured at densities ranging from 1 to 100 ÃÂ 106 cells/ml (200,000 to 20 ÃÂ 106 cells per well in flat-bottomed 96-well plates) with 20 pg/cell MSU crystals, 100 ng/ml PMA (MilliporeSigma), 5 ÃÂµg/ml ionomycin (Iono; MilliporeSigma), 1 ÃÂµM pyocyanin (MilliporeSigma), 15 ÃÂµM nigericin (NIG; InvivoGen, San Diego, CA, USA), 2.5 ÃÂµg/ml LPS from Klebsiella pneumoniae (MilliporeSigma), or without stimulus in HBSS medium containing 24 mM bicarbonate and 1 mM calcium at pH 7.4.
Techniques: Incubation, Microscopy, Staining, Marker, Cell Culture, Imaging