Ac yvad cmk yvad

Unless stated otherwise, THP-1 cells were seeded at a concentration of 1 × 10⁶/ml in ITS-RPMI media and were subjected to the four cell death stimuli as follows: To induce primary necrosis, cells were incubated at 37 °C for 3.25 h followed by incubation at 56 °C for 45 min (total incubation time 4 h); To induce secondary necrosis, cells were UV irradiated (150 mJ/cm²) using Stratagene UV Stratalinker 1800 (Agilent Technologies, CA) followed by incubation at 37 °C for 24 h; To induce pyroptosis, cells were primed with 1 μg/ml LPS (InVivogen, San Diego, CA) and incubated at 37 °C for 3 h. Cells were then treated with 10 μM nigericin (Sigma-Aldrich, St Louis, MO) for an additional 1 h (total incubation time 4 h); To induce apoptosis, cells were UV irradiated (150 mJ/cm²) using Stratagene UV Stratalinker 1800 followed by incubation at 37 °C for 4 h. Untreated control samples were incubated for either 4 h or 24 h at 37 °C. In all experiments involving UV irradiation, plastic covers of culture vessels were removed prior to irradiation to expose cells. In certain experiments, cells were pre-treated for 1 h with 50 μM Q-VD-OPh (Q-VD) or Ac-YVAD-cmk (YVAD) (Sigma-Aldrich).

Purified eicosapentaenoic acid and docosahexaenoic acid, palmitic acid, and oleic acid were obtained from Sigma Aldrich. Epanova® (omega-3-carboxylic acids [OM-3 CA]) was supplied by AstraZeneca. Over-the-counter omega-3-triglyceride supplements (Nordic Naturals) were purchased from Amazon. Safflower oil was purchased from a commercial grocer. Colchicine, indomethacin, and Ac-YVAD-cmk (YVAD) were purchased from Sigma Chemical. MSU, cholesterol, and calcium pyrophosphate crystals were made according to established protocols^{31 (link),32 (link)}. Crystals were tested and found to be free of endotoxins using PYROGENT™–5000 Kinetic Turbidimetric Limulus Amebocyte Lysate assays (Lonza). Cell-culture supernatant and pouch exudates were analysed for IL-1β content with electrochemiluminescence enzyme-linked immunosorbent assay (ELISA) plates (Mesoscale). Prostaglandin E₂ (PGE₂) content was analysed using a competitive enzyme immunoassay (R&D Systems).