*2.6. Isolation of Human Neutrophils and NETs Release*

Human neutrophils were isolated by density gradient centrifugation and hypotonic lysis [23]. Cells were adjusted to 2 × 10<sup>6</sup> /mL in RPMI-1640, seeded onto 8-well IbiTreat μ-slides (Ibidi #80826), 0.3 mL per well, and allowed to adhere for 15 min. PMNs were VitC loaded by incubating for 1 h with 0.3 mM or 3 mM buffered ascorbic acid (Mylan Institutional LLC, Rockville, IL, USA). Neutrophils were stimulated with 50 nM PMA for three hours at 37 °C. Neutrophil conditioned media were centrifuged at 400× *g* for 5 min and the supernatants used for quantification of cf-DNA [24].
