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.
Activation of THP-1 cells by uric acid:1. THP-1 cells are cultured in RPMI 1640 supplemented with 10% heat inactivated fetal bovineserum (FCS), 50uM β-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 50°C
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:Short-term Storage: +4°CLong-term Storage: -20°CStable for at least 1 year after receipt when stored at -20°C.
仕様
Size：10mg
Grade：Highly Purified
Form：Supplied as a liquid in PBS. Ready-to-use solution and is completely sterile and endotoxin-free.
