Neutrophil Activation Kinetics with Anakinra

For all assays, neutrophils were resuspended in Gibco RPMI 1640 media (Thermo Fisher, Waltham, MA, USA) containing 1% BSA (Sigma-Aldrich, Saint Louis, MO, USA). For the MPO and calcium measurements, the cells were seeded into white flat-bottom 96-well assay plates (Corning Incorporated, New York, NY, USA) at a density of 2 × 10⁵ cells per well. Supernatant sample generation for NE and cfDNA assays was done by seeding cells in a 24-well culture plate (Corning Incorporated, New York, NY, USA), at a concentration of 1 × 10⁶ cells per well. Cells were treated with concentrations of 100 ng/ml, 100 µg/ml and 1 mg/ml anakinra (Swedish Orphan Biovitrum AB, Stockholm, Sweden) at 60, 30, and 10 minutes prior, at the time of and 30 and 60 minutes after stimulation with 100 nM PMA or 5 µg/ml LPS (E.coli O55:B5) as indicated by other studies [25 (link), 27 (link)-29 (link)]. Stimulation was done for 3 hours.