Journal: Nutrients
Article Title: Zinc Supplementation Modulates NETs Release and Neutrophils’ Degranulation
doi: 10.3390/nu13010051
Figure Lengend Snippet: Zinc inhibits NETs release in neutrophils isolated from mice. Mice were sacrificed, and bone marrow neutrophils were isolated. ( a , b ) Neutrophils were stimulated to release NETs with 4 µM CI, 10 µM PAF or 20 µg/mL LPS E. coli . NETs release was ( a ) measured fluorometrically and ( b ) analyzed by fluorescent microscopy. ( c ) Neutrophils were incubated with 4 µg/mL DHR 123 and stimulated with 100 nM PMA. ROS production was measured fluorometrically. ( d ) Cells were incubated with E. coli bioparticles, and the percentage of cells phagocyting bacteria was analyzed by flow cytometry. ( e ) Neutrophils were primed with GM-CSF, incubated with cytochalasin B, C5a and labeled with anti-CD63. Data are shown as means ( a , d , e ) + SEM; (c) ± SEM; ( a , e ) with individual values. One-way ANOVA with post hoc ( a ) Bonferroni’s test, ( d ) Holm–Sidak’s test; ( e ) Dunn’s test. Two-way ANOVA with post hoc ( c ) Dunnett’s test. ( a , b ) n = 6, ( c ) n = 4, ( d ) n = 7, ( e ) n = 5; * ( p ≤ 0.05), ** ( p ≤ 0.01), **** ( p ≤ 0.0001). NE: neutrophil elastase.
Article Snippet: To analyze neutrophils degranulation, 5 × 10 5 of human cells were primed with 25 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF; Ref. no: 300-03, Peprotech, London, UK) for 15 min, incubated with 5 µg/mL cytochalasin B from Drechslera dematioidea (Ref. no: C6267, Sigma-Aldrich, St. Louis, MO, USA) for 5 min, followed by incubation with 10 nM of human C5a (Ref. no: HC2101, Hycult Biotech, Uden, The Netherlands) for 15 min. After this, the reaction was stopped with ice-cold PBS.
Techniques: Isolation, Microscopy, Incubation, Flow Cytometry, Labeling