Rabbit polyclonal anti cith3

Tetraethyl thiuram (TTD), aristolochic acid (AA), silymarin (SLN), phorbol 12-myristate 13-acetate (PMA), porcine skin gelatin, collagen-I/IV, laminin, fibronectin, Hoechst stain, DNase 1, ficoll-paque, dextran and phosphatase inhibitor cocktail were obtained from Sigma-Aldrich (Bangalore, India). BSA, ethanol, dimethyl sulfoxide (DMSO; HPLC grade), Tween-20 and Hank’s balanced salt solution (HBSS) were purchased from HiMediaLaboratories, Pvt. Ltd. (Mumbai, India). SCH79797 (PAR-1 antagonist) and GB-83 (PAR-2 antagonist) were purchased from Cayman Chemicals (Michigan, USA). U0126 (MEK 1/2 inhibitor), antibodies against p-ERK, β-actin and cell lysis buffer were purchased from Cell Signaling Technology (Massachusetts, USA). HRP tagged anti-rabbit IgG and anti-mouse IgG were procured from Jackson ImmunoResearch (Philadelphia, USA). The rabbit polyclonal anti-citH3, rabbit polyclonal anti-H3, mouse monoclonal anti-myeloperoxidase (anti-MPO) and anti-PAD4 were obtained from Abcam (Cambridge, UK). All other chemicals and reagents used in this study are analytical grade.

Colon tissue was snap-frozen and homogenized in RIPA buffer (Sangon Biotech, Shanghai, China) containing protease inhibitors on ice. The homogenate was centrifuged at 12,000 rpm for 10 min at 4°C and the total protein was quantified using a BCA (Beyotime Biotechnology, Shanghai, China) assay. Equal amounts of proteins were mixed with loading buffer, loaded on gradient gels (12% SDS-PAGE), and then transferred to a PVDF membrane. The membrane was blocked with 5% milk in TBST and incubated with primary antibodies at 4°C overnight as listed: rabbit polyclonal anti-Cit-H3 (1:1000, ABCAm); rabbit polyclonal anti-GAPDH (1:2000, Servicebio); rabbit polyclonal anti-PAD4 (1:5000, Proteintech); rabbit polyclonal anti-MPO (1:3000, Proteintech); rabbit polyclonal anti-occludin (1:3000, Proteintech). After washing, the blot was finally incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5000, Proteintech) for 1h at room temperature. Bands were visualized using a chemiluminescent substrate (Sangon Biotech) and were quantified based on the endogenous GAPDH level using ImageJ software.

10⁵ isolated human or mouse neutrophils were seeded on glass coverslips in tissue-culture plates and incubated with PMA (100 ng/ml, Sigma-Aldrich), Ionomycin (1 µM, Sigma-Aldrich), or 5 × 10³C. albicans preformed hyphae for 2.5 h at 37°C. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.01% TritonX-100 for 10 min, and blocked with 5% donkey serum for 30 min at room temperature. Cells were stained with goat polyclonal anti-MPO (R&D systems), rabbit polyclonal anti-CitH3 (Abcam), and combined rat anti-S100A8 and anti-S100A9 (clone 10 and clone 46 respectively, kindly provided by C. Urban, Umeå, Sweden). Secondary antibodies were donkey anti-goat IgG (Jackson ImmunoResearch), goat anti-rat IgG (Abcam), and goat anti-rabbit IgG (Jackson ImmunoResearch). The PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich) was used to stain lipids of the neutrophil plasma membranes. DNA was stained with 4′,6′-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich) and NETs were visualized using a Leica SP8 inverse confocal microscope and analyzed with FIJI software.