Human serum albumin (hsa)

Biotin-labeled proteins were prepared by incubating 5 mg/ml BSA (Iwai Chemicals), human serum albumin (FUJIFILM Wako Pure Chemical, Inc), transferrin (FUJIFILM Wako Pure Chemical, Inc), and IgG (FUJIFILM Wako Pure Chemical, Inc) with a 10-fold molar Bt-PE-maleimide (Dojindo Laboratories) in PBS (10 mM sodium phosphate buffer (pH 7.4)) at 25 °C for 16 h. After incubation, the aliquots were dialyzed against PBS. The polyphenol-modified proteins were prepared by incubating 1 mg/ml proteins or biotinylated proteins with 2.5 mM polyphenols in PBS containing 10% dimethyl sulfoxide for 72 h at 37 °C under atmospheric oxygen. After incubation, the reactants were collected and dialyzed with PBS. The protein concentrations were measured by a bicinchoninic acid assay (Nacalai Tesque, Inc). Bt-AET was synthesized according to previous studies (28 ). The polyphenol-modified amino acid analogs were prepared upon incubation of 1 mM Bt-APA (Thermo Fisher Scientific, Inc) or Bt-AET with 2.5 mM polyphenols in PBS containing 10% dimethyl sulfoxide for 24 h at 37 °C under atmospheric oxygen. The oxidized Bt-APA was prepared according to a previous report (2 (link)).

Extracellular DNA was stained with 5 μm SYTOX Green (Life Technologies, Carlsbad, CA, USA), a fluorescent membrane‐impermeable DNA dye. Fluorescence was quantified using a microplate reader equipped with filters to detect excitation/emission maxima of 504/523 nm (EnSpire; PerkinElmer, Waltham, MA, USA). When indicated, neutrophils were stimulated with 20 or 200 nm PMA, or cultured in the presence of 100 U·mL⁻¹ DNase I (TaKaRa, Osaka, Japan), 40 mg·mL⁻¹ bovine serum albumin (BSA; Wako Pure Chemical Industries, Osaka, Japan), 40 mg·mL⁻¹ human serum albumin (Wako), heat‐treated (complement‐inactivated) serum, protein G/A sepharose‐treated (immunoglobulin‐depleted) serum, 10 mm EDTA, or 5 or 20 mm N‐acetyl‐l‐cysteine (NAC; Wako).