Af3667

After stimulation, neutrophils were fixed with 4% paraformaldehyde. Protein staining was performed using a rabbit polyclonal anti-citH3 antibody (1:200, ab5103, Abcam), a mouse monoclonal anti-NE antibody (1:50, sc-55549, Santa Cruz), and a goat monoclonal anti-MPO antibody (1:200, AF3667, R&D) overnight at 4 °C. After three washes, appropriate fluorochrome-conjugated secondary antibodies (1:200, Alexa Fluor 594-conjugated rabbit anti-mouse IgG, 33912ES60; 1:200, Alexa Fluor 488-conjugated goat Anti-rabbit IgG, 33106ES60; 1:200, Alexa Fluor 647-conjugated rabbit anti-goat IgG, 33713ES60; all from YEASEN, Shanghai, China) were applied for 1 h incubation at room temperature. DNA was stained with Hoechst 33342 (1:2000, H3570, Invitrogen) for 5 min. After three washes, images were obtained using FV3000 confocal system (Olympus).
For quantification, total extracellular DNA generated by cultured neutrophils was digested with 30 mU/mL micrococcal nuclease (MNase, Thermo Fisher Scientific) for 10 min at 37 °C, and then stopped with 5 mM EDTA. Cell-free DNA and MPO-DNA complex in the supernatants was quantified by PicoGreen. For BMDNs, NET formation was confirmed by visualization using 0.3 μM SYTOX green (S7020, Invitrogen, USA) and images were captured using an Olympus microscope (IX73).

Smarcad1 immunofluorescent (IF) staining was performed as described in [87 (link)] with the following antibodies: primary anti-Smarcad1 (Sigma-Aldrich HPA016737, 1.5 μg/ml) and secondary anti-rabbit AlexaFluor 568 (Invitrogen A11036, 2 μg/ml). MPO, Adamts1, Adamts5, and Bmp1 IF stainings were performed, and MPO-positive cells quantified as described in [88 (link)] with the following antibodies: primary anti-MPO (R&D AF3667, 5 μg/ml), primary anti-Adamts1 (Abcam ab39194, 1 μg/ml), primary anti-Adamts5 (Abcam ab246975, 0.5 μg/ml), primary anti-Bmp1 (Abcam ab38953, 2 μg/ml), and secondary anti-goat AlexaFluor 488 (Invitrogen A11055, 10 μg/ml) and anti-rabbit AlexaFluor 568 (Invitrogen A11036, 20 μg/ml). Confocal imaging was performed on a Zeiss 780 confocal microscope with a × 20 Plan Apo air objective at optimal resolution settings with 2× line averaging. Contrast enhancement with minor pixel saturation was performed with FIJI [89 (link)]. MPO-staining was performed as 2–4 technical replicates (different intestinal positions within the same sample) that were measured and averaged per sample. The following are the average total imaged per sample: 0.47 mm² epithelial area and 0.30 mm² sub-epithelial area. Imaging areas were measured with FIJI, and positive cells counted manually after background correction.

Immunofluorescent stainings were prepared and confocal imaging performed as described^{48 (link)} with primary Myeloperoxidase (MPO)-antibody (R&D AF3667, 5 µg/ml) incubated 2 h at room temperature and secondary anti-goat AlexaFluor 488 (Invitrogen A11055, 10 µg/ml) incubated at 4 °C over night. Permeabilization was performed for 4 min in 0.3% TritonX/PBS. 2% BSA/2% donkey serum/PBS was used as blocking solution. No unmasking was performed. Imaging was performed on a Zeiss 780 confocal microscope with a 20 × Plan Apo air objective at optimal resolution settings with 2 × line averaging. 5 × 2 µm optical stacks were processed as maximum intensity overlays (optical thickness 8 µm. Contrast enhancement (thresholding background signal) were performed with FIJI^{47 (link)}. Three technical replicates (different intestinal positions within the same sample) were measured and averaged per sample. Average total imaged crypt area per sample: 0.16 mm², villus area 0.18 mm². Crypt and villus areas were measured with FIJI, positive cells counted manually after background correction.