| NLRP3/NALP3 inflammasome activator. Specially crystallized and tested for biological activity. Stimulates the caspase-1 activating NLRP3/NALP3 inflammasome, resulting in the production of active IL-1beta and IL-18. Synonyms: 7,9-Dihydro-1H-Purine-2,6,8(3H)-trione, 1H-Purine-2,6,8-triol, 2,6,8-Trihydroxypurine, 2,6,8-Trioxopurine, 2,6,8-Trioxypurine, Lithic Acid, NSC 3975, Purine-2,6,8(1H,3H,9H)-trione CAS No: 1198-77-2 Molecular Formula: C5H3N4O3Na Molecular Weight: 190.1 Appearance: Solid crystals Endotoxin: 0.001EU/ug purified protein (LAL method) Solubility: Not soluble in PBS. Reconstitute 2mg of MSU in 100ul 1M sodium hydroxide to obtain a 20mg/ml suspension. Activation of THP-1 cells by uric acid: 1. THP-1 cells are cultured in RPMI 1640 supplemented with 10% heat inactivated fetal bovine serum (FCS), 50uM beta-mercaptoethanol, 2mM L-glutamine and penicillin/streptomycin. THP-1 cells are grown in suspension to a density of 1.5x10e6 cells/ml in tissue culture flasks. 2. Treat THP-1 cells for 3 hours with 0.5uM PMA the day before stimulation. 3. Wash cells with PBS and plate them in a 12-well plate at a density of 6x10e5 cells/well in RPMI medium. 4. The next day, wash cells with PBS and add 500ul Optimem (Invitrogen). 5. Stimulate the cells for 6 hours by addition of 50-300ug/ml of the suspension of uric acid (vortex the suspension just before use). 6. As a positive control, 5mM of ATP is incubated for 6 hours. Detection of the mature Caspase-1 in the supernatant of uric acid-activated THP-1 cells: 1. Collect the supernatant of each well (500ul). Add 500ul methanol and 125ul chloroform. 2. Vortex and centrifuge for 5 min at 13,000 rpm. 3. Remove the upper phase. 4. Add 500ul methanol and centrifuge for 5 min at 13,000 rpm. 5. Remove the supernatant, dry the pellet for 5 min at 50C. 6. Add 60ul of 1X loading buffer and DTT (final concentration of 0.1M). 7. Mix and boil before loading on a 15% SDS-gel and transfer onto nitrocellulose membrane. The mature Caspase-1 is detected using the Caspase-1 (p20) (human), mAb (Bally-1) antibody (Cat No 044627). Storage and Stability: May be stored at 4C for short-term only. Long-term storage is recommended at -20C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial prior to removing the cap. |
