Sml 1779

Bone marrow-derived macrophages (BMDMs) were preincubated with LPS (20 ng/mL) for 3 h and then incubated with 2 mM ATP (Sigma-Aldrich, GE27-2056-01) or 10 μg/mL Nigericin (Sigma-Aldrich, SML-1779) for 1 h. To assess inflammasome activation in neutrophils, control and Myl-Abc^dko neutrophils were incubated with a conditioned medium from control macrophages treated with LPS or LPS + ATP as described in the in vitro NET assay section above. Net IL-1β secretion from neutrophils was calculated by subtracting IL-1β concentration before incubation with macrophage-conditioned medium from the IL-1β concentration after incubation with macrophage-conditioned medium. IL-1β secretion into the media was measured using ELISA (R&D Systems; DY401). Data were normalized to a protein concentration of neutrophil cell lysates. IL-1β cleavage in neutrophil cell lysates was assessed by western blot using a primary antibody from Abcam (ab9722) and an anti-rabbit secondary antibody from Cell Signaling (7074).

CD8⁺ T cells from healthy donors were isolated and transfected with siRNA or mRNA as described above. Cells were harvested at a density of 1 × 10⁶/mL and labeled with Indo-1 AM (5 μmol/L; catalog no. 565879, BD Pharmingen) at 37 °C for 30 minutes. Cells were washed with complete RPMI and incubated in complete RPMI for another 30 minutes at 37 °C. Finally, cells were suspended and kept in 37°C water bath before acquisition on an LSR II cytometer (BD Biosciences) running FACSDiva v6.1.3 software (RRID:SCR_001456). After baseline acquisition for 1 minute, the analogue of nicotinic acid, adenine dinucleotide phosphate (NAADP-AM; 10 μmol/L; catalog no. 21000, AAT Bioquest) was added and sample collection immediately resumed. In addition, we used trans-Ned-19 (an inhibitor of NAADP pathway; 10 μmol/L, Enzo Life Sciences, catalog no. ALX-270–503-M001) or Nigericin (an ionophore agent that depletes acidic Ca²⁺ stores; 10 μmol/L, Sigma, catalog no. SML1779) in culture medium 30 minutes before the addition of NAADP-AM to test effects on calcium release at basal level and induced by NAADP-AM. Flow data was analyzed by FlowJo, using the kinetic analyzer function (FlowJo, LLC).