N formyl methionyl phenylalanine fmlp

Anti-human CD66b, Annexin-V-Alexa647 and Annexin-V-FITC were purchased from BioLegend (San Diego, CA, USA). Calcein Blue AM was from Invitrogen (Waltham, MA, USA). Antibodies against MPO were from Dako (A0398). Ficoll-Paque Plus was from GE Biosciences (Baie d’Urfé, QC, Canada); endotoxin-free (<2 pg/mL) RPMI 1640 was from Wisent (St-Bruno, QC, Canada). Recombinant human cytokines were from R&D Systems (Minneapolis, MN, USA). N-formyl-methionyl-phenylalanine (fMLP) and phenylmethanesulphonyl fluoride (PMSF) were from Sigma (St. Louis, MO, USA). All inhibitors, antagonists, and fluorescent probes were purchased through Cedarlane Labs (Mississauga, ON, Canada). PlaNET reagents (fluorescent chromatin-binding polymers) are no longer available from Immune Biosolutions or other suppliers; we therefore employed a close equivalent: fluorescent, 50-nm carboxylate microspheres (# 16661-10) from Polysciences Inc. (Warrington, PA, USA). These microspheres are referred to as PlaNET reagents throughout this study, since the name is neither registered as a trademark, nor under copyright. All other reagents were of the highest available grade, and all buffers and solutions were prepared using pyrogen-free, clinical grade water.

Antibodies against myeloperoxidase (A0398) were from Dako/Agilent (Mississauga, ON, Canada); antibodies against citrullinated histone H3 were from Abcam (Ab5103); phospho antibodies were from Cell Signaling (Beverly, MA, USA). Ficoll-Paque Plus was from GE Biosciences (Baie d'Urfé, Qc, Canada); endotoxin-free (< 2 pg/ml) RPMI 1640 was from Wisent (St-Bruno, Qc, Canada). Recombinant human cytokines were from R&D Systems (Minneapolis, MN, USA). Actinomycin D, cycloheximide, N-formyl-methionyl-phenylalanine (fMLP), and phenylmethanesulphonyl fluoride (PMSF) were from Sigma (St. Louis, MO, USA). Kinase inhibitors and fluorescent probes were all purchased through Cedarlane Labs (Mississauga, Canada). PlaNET reagents, fluorescent chromatin-binding polymers, were from Sunshine Antibodies (https://sunshineantibodies.com/planet-001.html).

Neutrophils were purified from bone marrow as described previously.10 Briefly, bone marrow‐derived neutrophils were harvested using 62% Percoll. For the actin polymerization assay, purified neutrophils (4 × 10⁵ cells) were suspended in HBSS (Nacalai Tesque) containing 0.2% bovine serum albumin (Sigma Aldrich) and allowed to adhere to fibronectin‐coated coverslips (Neuvitro) for 15 minutes at 37°C in a 5% CO₂ incubator. Neutrophils were treated with either 1000, 100, 10, or 1 nmol/L of commercially available Cayman (±)17,18‐EpETE, BM‐3 17(S),18(R)‐EpETE, 18‐HEPE (Cayman Chemical), RvE1 (Cayman Chemical), or 0.03% (vol/vol) ethanol (vehicle control) for 15 minutes and then stimulated with 1 μmol/L N‐formyl‐methionyl‐phenylalanine (fMLP; Sigma Aldrich) or 100 nmol/L leukotriene B₄ (LTB₄; Cayman Chemical) for 2 minutes at 37°C in a 5% CO₂ incubator. Neutrophils were fixed in 4% paraformaldehyde (Nacalai Tesque), permeabilized using 0.5% (vol/vol) Triton X‐100 (Nacalai Tesque) in PBS, and stained with 100 nmol/L Acti‐stain 488–phalloidin (Cytoskeleton) for 30 minutes at room temperature. Finally, cell nuclei were stained by incubating neutrophils with 4ʹ,6‐diamidino‐2‐phenylindole for 30 seconds at room temperature. Images were obtained with Leica TCS SP8 confocal microscopy (Leica Microsystems).