quant-ittm picogreen® dsdna reagent and kits  (Thermo Fisher)

Journal: International Journal of Molecular Sciences

Article Title: Neutrophil Extracellular Trap Formation Model Induced by Monosodium Urate and Phorbol Myristate Acetate: Involvement in MAPK Signaling Pathways

doi: 10.3390/ijms26010143

Figure Lengend Snippet: MSU and PMA induces NET formation: ( A ) The morphological changes of cells treated with MSU and/or PMA were observed under light microscope. Scale bars, 100 μm. ( B ) Human neutrophils were seeded in 12-well plates containing L-polylysine slides. After treatment with MSU and/or PMA, the cells were fixed 4% paraformaldehyde. The formation of NETs was observed using Sytox Green dead cell nucleic acid dye (green) and Hoechst 33342 dye (blue). Scale bars, 50 μm. ( C ) The generation of NETs was quantified by Sytox Green dye at Ex/Em: 502/525 nm using a fluorescence microplate reader, n = 3. ( D ) Human neutrophils (2 × 10 5 cells/well) were seeded in 12-well plates containing L-polylysine slides. Triton X-100 was permeabilized and 5% BSA was blocked. Then, samples were stained with these primary antibodies: rabbit Histone H3 (citrulline R2 + R8 + R17) (CitH3) antibody (1:400, green), goat MPO antibody (1:200, red), mouse NE antibody (1:200, pink), they incubated in a wet box at 4 °C overnight. Secondary antibodies were then blocked for 1 h at room temperature and DAPI (blue) for 10 min. Compared to unstimulated neutrophils, MSU and/or PMA-stimulated cells show membrane rupture and release of cellular contents (such as MPO and NE), appearing as filamentous structures, with some histones undergoing citrullination. Scale bars, 20 μm. ( E ) Neutrophils (1 × 10 6 cells/mL) were seeded in a 6-well plate. After various treatments, the supernatants were collected. The supernatants were then centrifuged, and the resulting supernatants were mixed with PicoGreen reagent and incubated at room temperature in the dark for 5 min. The fluorescence intensity was measured at Ex/Em: 480/520 nm, n = 4. The data were presented as mean ± SEM with ** p < 0.01 vs. Control group. MSU, monosodium urate; PMA, phorbol 12-myristate 13-acetate; NET, neutrophil extracellular trap; CitH3, citrulline Histone H3; MPO, myeloperoxidase; NE, neutrophil elastase.

Article Snippet: Quant-iTTM PicoGreen ® dsDNA Reagent and Kits were from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Light Microscopy, Fluorescence, Staining, Incubation, Membrane, Control

Journal: International Journal of Molecular Sciences

Article Title: Neutrophil Extracellular Trap Formation Model Induced by Monosodium Urate and Phorbol Myristate Acetate: Involvement in MAPK Signaling Pathways

doi: 10.3390/ijms26010143

Figure Lengend Snippet: The formation of NETs can be prevented by inhibitors of the MAPK signaling pathway: ( A ) The morphological changes of cells treated with different selective inhibitors were observed under a light microscope. Scale bars, 100 μm. ( B ) Sytox Green dead cell nucleic acid dye (green) and Hoechst 33342 dye (blue) were used to observe the intervention of different inhibitors on NET formation. Scale bars, 50 μm. ( C ) The generation of NETs was quantified by Sytox Green dye at Ex/Em: 502/525 nm using a fluorescence microplate reader. ( D ) The cell supernatant dsDNA was measured with PicoGreen staining followed by a fluorescent microplate reader, n = 4. ( E ) Observation of NET release by laser confocal microscopy. Scale bars, 20 μm. DPI is a NOX inhibitor, Salirasib is a Ras inhibitor, Vemurafenib is a Raf inhibitor, PD98059 is an ERK inhibitor, SB203580 is a p38 MAPK inhibitor, Alvelestat is a NE inhibitor. (The same below.) The data were presented as mean ± SEM with # p < 0.05 or ## p < 0.01 vs. MSU + PMA group. DPI, diphenyleneiodonium chloride; NOX, NADPH oxidase.

Article Snippet: Quant-iTTM PicoGreen ® dsDNA Reagent and Kits were from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Light Microscopy, Fluorescence, Staining, Confocal Microscopy