1640 glutamax 1

The human gastric epithelial cell line AGS (ATCC^® CRL-1739) was cultured in RPMI medium 1640 Glutamax I (Gibco, Invitrogen, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, HyClone, Thermo Fisher Scientific, Inc, UK) and 1% (v/v) Penicillin/Streptomycin (Gibco) at 37°C in a humidified 5% CO₂ atmosphere. Cells were seeded at 1.6 x 10⁵ cells/well in a 96-well plate. After overnight, media were changed and cells were incubated with predetermined concentrations (0.4 μM to 2 μM) of Cy3_HP_ LNA/2OMe _PS probe diluted in vehicle (0.5 M urea and 900 mM NaCl) or only with the vehicle, for 24 h. Cell viability was assessed by CellTilter 96^® Aqueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI), according to the manufacturer’s instructions. Untreated cells served as a negative control. The experiments were performed in triplicate.

Lenti-X 293T cells (Takara) were cultured at 0.5–1 × 10⁶ cells/ml in Dulbecco’s modified Eagle’s Medium (DMEM) GlutaMax-I supplemented with 1X penicillin/streptomycin (PS) and 10% fetal bovine serum (FBS) (Gibco). NLRP3-deficient U937 cells have been previously described (Lagrange et al., 2018 (link)). U937 and reconstituted U937 NLRP3-deficient cells were cultured at 0.25–1 × 10⁶ cells/ml in Roswell Park Memorial Institute (RPMI) 1640 GlutaMax-I supplemented with 1X PS and 10% FBS (Gibco). Monocytes were purified from 10 to 20 ml of blood samples (drawn the day before in heparin tubes) using EasySep Direct Human Monocyte Isolation kit (StemCell) and cultured at 0.07–0.15 × 10⁶ cells/ml in RPMI-1640 GlutaMax-I supplemented with 1X PS and 10% FBS (Gibco). The purity of the monocyte fractions was assessed by anti-CD45-APCR700 and anti-CD14-PE-CF594 (BD Horizon) staining and FACS analysis, and ranged between 66 and 82% CD45+/CD14+ (gated on total cells) and 81–95% (gated on CD45%).

NCI-H69 human small-cell lung carcinoma cells (ECACC) were sub-cultured once a week at a concentration of 180,000 cells/mL (T175 culture flask, 20 mL), and fresh medium was added on day four (30 mL). Cells were cultured up to 20 passages in a humidified atmosphere (37 °C, 5% CO₂) in RPMI medium 1640 + GlutaMAX-I (Gibco, Breda, The Netherlands) supplemented with 10% (v/v) fetal bovine serum (Gibco) and 5% (v/v) penicillin–streptomycin (Merck Life Science NV, Amsterdam, The Netherlands).