Monosodium Urate (MSU), crystals
|Biochemicals||Storage: -20°CShipping: RT|
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.
Not soluble in PBS. Reconstitute 2mg of MSU in 100ul PBS 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 -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:
May be stored at 4ºC for short-term only. Long-term storage is recommended at -20°C. Stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial prior to removing the cap.
C5H3N4O3 . Na
Form: Solid crystals, endotoxin-free.
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.