S7 nuclease

Isolated neutrophils were seeded at a concentration of 2 × 10⁶ cells/ml, stimulated with 50 nM PMA, washed, digested with S7 nuclease (Cayman Chemicals) for 20 min, centrifuged and stored at −20°C. Neutrophil elastase, myeloperoxidase (both from Abcam), LL37 (Hycult Biotech, Wayne, NJ, United States) and DNA-histone associated complex (Sigma-Aldrich, St. Luis, United States) concentrations were evaluated by ELISA. The DNase I concentration in the human serum was also determined by ELISA (LifeSpan BioSciences, Seattle, WA, United States).

dHL-60 cells were cultured in RPMI1640 containing 1% BSA, 1% antibiotics, and 10 mM HEPES (HO887, Sigma-Aldrich). To stimulate NET formation in dHL-60 cells, 100, 200, and 400 nM phorbol myristate acetate (PMA; 601010, Cayman, Ann Arbor, MI) or serum from HD patients was used. As a control, serum from HVs was used. HL-60 cells differentiated for 6 days by ATRA were seeded at 5 × 10⁵ cells in a 24-well plate and incubated for 17 h. Next, cells were treated with 150 U/mL S7 nuclease (601010, Cayman) for 1 h and the supernatant was transferred to a microtube (MCT-150-C, AXYGEN, Corning, NY). Nuclease was inactivated by adding 10 mM EDTA to the supernatant and centrifugation at 500g for 5 min. The supernatant was transferred to a new microtube and stored at – 20 °C until analysis.

NET samples were produced according to the instructions of the NETosis Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). Briefly, 3.6 × 10⁵ cells per well were plated in clear 48-well plates and cultured with endpoints as mentioned above followed by two washing steps with NET Assay Buffer (RPMI 1640 containing 1% BSA and 1 mM CaCl₂). Subsequently, NETs were disrupted by S7 nuclease (Cayman Chemical, Ann Arbor, MI, USA) and collected. Samples were stored at -20°C for up to two weeks.