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
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
Molecular Formula
C5H3N4O3Na
Appearance
Supplied as a liquid in PBS. Ready-to-use solution and is completely sterile and endotoxin-free.
Storage and Stability
May be stored at RT for short-term only. Long-term storage is recommended at -20°C. For maximum recovery of product, centrifuge the original vial prior to removing the cap.
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 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°C Long-term Storage: -20°C Stable for at least 1 year after receipt when stored at -20°C.
Important Note
This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
Toxicity and Hazards
All products should be handled by qualified personnel only, trained in laboratory procedures.
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