Journal: Cells

Article Title: Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study

doi: 10.3390/cells12172199

Figure Lengend Snippet: Scheme of experiment using human donor neutrophils exposed to A23187 and PMA. Stage 1: Isolation of neutrophils. Stage 2: Activation of neutrophils by A23187/PMA and exposure to CO 2 . Colocalization of decondensed chromatin with myeloperoxidase after neutrophil stimulation with PMA and A23187. Blue, staining of chromatin with DAPI; green, staining of MPO with FITC-conjugated anti-MPO monoclonal antibodies. Scale bar: 25 µm. Stage 3: Acquisition of AFM and fluorescence images.

Article Snippet: Nonspecific binding was reduced by preincubation of cells with blocking buffer (including human immunoglobulins) for 20 min. Immunofluorescence staining was performed with FITC-labeled monoclonal mouse anti-human MPO priming antibodies and neutrophil DNA was stained with DAPI (10 µM) for 10 min. For CLSM and widefield microscopy, cells were permeabilized with TritonX-100 0.05% (Sigma, St. Louis, MO, USA) for 15 min, then washed and blocked with 3% BSA.

Techniques: Isolation, Activation Assay, Staining, Fluorescence

Journal: RMD Open

Article Title: New pro-inflammatory cytokine IL-40 is produced by activated neutrophils and plays a role in the early stages of seropositive rheumatoid arthritis

doi: 10.1136/rmdopen-2022-002894

Figure Lengend Snippet: Serum levels of neutrophil extracellular traps markers (MPO, PR3, NE) are up-regulated in ERA patients (n=60) at baseline compared with HC (n=60) (A). Serum IL-40 significantly correlates with the peripheral blood neutrophil count (B), with serum levels of PR3 (C), NE (D), MPO (E) (n=60) and with cfDNA on the border of statistical significance (F) (n=35). Horizontal lines represent the median. The association of IL-40 with variables was analysed using Spearman correlation. ****p<0.0001. cfDNA, cell-free DNA; ERA, early rheumatoid arthritis; HC, healthy controls; IL-40, interleukin 40; MPO, myeloperoxidase; NE, neutrophil elastase; ns, non-significant; PR3, proteinase 3.

Article Snippet: Slides were incubated with mouse anti-human myeloperoxidase (MPO) monoclonal antibody over night at 4°C (1:200, Invitrogen, Carlsbad, CA, USA) and C17orf99 rabbit anti-human polyclonal antibody (1:700, Sinobiological, Eschborn, Germany) for 3 hours at RT.

Techniques:

Journal: RMD Open

Article Title: New pro-inflammatory cytokine IL-40 is produced by activated neutrophils and plays a role in the early stages of seropositive rheumatoid arthritis

doi: 10.1136/rmdopen-2022-002894

Figure Lengend Snippet: Immunofluorescence staining of IL-40 release during externalisation of neutrophil extracellular traps (NETosis) in ERA neutrophils (n=4). The representative images show fluorescent staining of DNA (blue), wheat germ agglutinin that binds to the plasma membrane (WGA, magenta), and immunofluorescent staining of neutrophil marker myeloperoxidase (MPO, red) and IL-40 (green) and their merged images. In non-NETotic neutrophils (ctrl), IL-40 partially overlaps with the intact plasmatic membrane (white arrow). A small proportion of IL-40 signal can also be observed in the cytoplasmic area (white arrow heads). IL-40 in the central part of the cell does not merge with MPO. PMA-treated neutrophils release decondensed chromatin that does not colocalise with IL-40 (green arrows). Similarly, DNA-appendant MPO does not merge with IL-40. There is a partial overlap in IL-40 signal and plasma membrane remnants (blue arrows). The images were obtained using confocal microscopy, MPO and IL-40 in detail of PMA-treated cells were obtained by STED microscopy. Scale bar: 8 µm. ctrl, unstimulated cells; ERA, early rheumatoid arthritis; IL-40, interleukin-40; MPO, myeloperoxidase; NETs, neutrophil extracellular traps; PMA, phorbol 12-myristate-13-acetate; STED, stimulated emission depletion; WGA, wheat germ agglutinin.

Article Snippet: Slides were incubated with mouse anti-human myeloperoxidase (MPO) monoclonal antibody over night at 4°C (1:200, Invitrogen, Carlsbad, CA, USA) and C17orf99 rabbit anti-human polyclonal antibody (1:700, Sinobiological, Eschborn, Germany) for 3 hours at RT.

Techniques: Immunofluorescence, Staining, Marker, Confocal Microscopy, Microscopy

Journal: RMD Open

Article Title: New pro-inflammatory cytokine IL-40 is produced by activated neutrophils and plays a role in the early stages of seropositive rheumatoid arthritis

doi: 10.1136/rmdopen-2022-002894

Figure Lengend Snippet: Recombinant IL-40 does not induce externalisation of neutrophil extracellular traps in ERA neutrophils (n=4) (A). The representative image shows fluorescent staining of DNA (blue), wheat germ agglutinin that binds to the plasma membrane (WGA, magenta) and immunofluorescent staining of neutrophil marker myeloperoxidase (MPO, red) and their merged images. The quantification of cfDNA (B) and MPO (C) release by IL-40 stimulated neutrophils further supports the abovementioned. The images were obtained using confocal microscopy. Scale bars: 2.9 µm for ctrl and IL-40-stimulated cells, 16.7 µm for PMA-stimulated cells. cfDNA, cell-free DNA; ctrl, unstimulated cells; ERA, early rheumatoid arthritis; IL-40, interleukin 40; MPO, myeloperoxidase; NETs, neutrophil extracellular traps; PMA, phorbol 12-myristate-13-acetate; WGA, wheat germ agglutinin.

Article Snippet: Slides were incubated with mouse anti-human myeloperoxidase (MPO) monoclonal antibody over night at 4°C (1:200, Invitrogen, Carlsbad, CA, USA) and C17orf99 rabbit anti-human polyclonal antibody (1:700, Sinobiological, Eschborn, Germany) for 3 hours at RT.

Techniques: Recombinant, Staining, Marker, Confocal Microscopy