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Monosodium urate (crystals)

NLRP3 inflammasome activator
ALX-400-047-M002 2 mg 95.00 USD
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NLRP3/NALP3 inflammasome activator. Specially crystallized and tested for biological activity.

Product Specification

Alternative Name:MSU (crystals), Uric Acid (crystals)
Formula:C5H3N4O3 . Na
Identity:Determined by IR.
Appearance:Solution may appear as a slightly hazy suspension.
Activity:Stimulates the caspase-1 activating NLRP3/NALP3 inflammasome, resulting in the production of active IL-1β and IL-18.
Solubility:Soluble in PBS or 1M NaOH (25 mg/ml).
Long Term Storage:-20°C
Handling:Vortex before use.
Protocol: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), 50μM β-mercaptoethanol, 2mM L-glutamine and penicillin/streptomycin. THP-1 cells are growN in& suspension to a density of 1.5x106 cells/ml in tissue culture flasks.
  2. Treat THP-1 cells for 3 hours with 0.5µM PMA (Prod. No. BML-PE160) the day before stimulation.
  3. Wash cells with PBS and plate them in a 12-well plate at a density of 6x105 cells/well in RPMI medium (this increases the phagocytic properties of the cells and induces a constitutive production of proIL-1β).
  4. The next day, wash cells with PBS and add 500µl Optimem (Invitrogen).
  5. Stimulate the cells for 6 hours by addition of 50-200µg/ml of the suspension of uric acid (vortex the suspension just before use).
  6. As a positive control, 5mM of ATP or 50µg/ml of LPS from E. coli, Serotype R515 (Re) (TLRgrade™) (liquid) (Prod. No. ALX-581-007) are incubated for 6 hours.

Detection of the mature IL-1β in the supernatant of uric acid-activated THP-1 cells:
  1. Collect the supernatant of each well (500µl). Add 500µl methanol and 125µl chloroform.
  2. Vortex and centrifuge for 5 min. at 13’000 rpm.
  3. Remove the upper phase.
  4. Add 500µl 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 60µl 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 IL-1β is detected using the cleaved IL-1β (Asp116) antibody.
→Note: Detection of IL-1β in the supernatant of uric acid-activated THP-1 cells can also be performed by ELISA.
→Note: Sonication of MSU improves its biological activity in
murine cells.
Monosodium urate (crystals) Western blot
Figure: Detection of the mature IL-1 in the supernatant of THP-1 cells by Western blot after treatment with Monosodium urate (crystals) (Prod. No. ALX-400-047). Method: THP-1 cells were treated with 10, 50 or 250µg of uric acid and with 50µg of LPS from E. coli, Serotype R515 (Re) (TLRgrade™) (liquid) (Prod. No. ALX-581-007) as mentioned in the protocol. The supernatant was collected, precipitated and a fraction was loaded in a 15% SDS-gel."
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400-047 Monosodium urate (crystals) Western blot

Product Literature References

Early endosome autoantigen 1 regulates IL-1β release upon caspase-1 activation independently of gasdermin D membrane permeabilization: A. Baroja-Mazo, et al.; Sci. Rep. 9, 5788 (2019), Abstract; Full Text
P2Y6 Receptor Signaling Pathway Mediates Inflammatory Responses Induced by Monosodium Urate Crystals: H. Uratsuji, et al.; J. Immunol. 188, 436 (2012), Abstract;
Inflammasome activation in NADPH oxidase defective mononuclear phagocytes from patients with chronic granulomatous disease: F. Meissner, et al.; Blood 116, 1570 (2010), Abstract; Full Text
Monosodium urate crystals in inflammation and immunity: Y. Shi, et al.; Immunol. Rev. 233, 203 (2010), Abstract;
Uric Acid-Driven Th17 Differentiation Requires Inflammasome-Derived IL-1 and IL-18: C. Conforti-Andreoni, et al.; J. Immunol. 187, 5842 (2010), Abstract;
Crystals of monosodium urate monohydrate enhance lipopolysaccharide-induced release of interleukin 1 beta by mononuclear cells through a caspase 1-mediated process: E.J. Giamarellos-Bourboulis, et al.; Ann. Rheum. Dis. 68, 273 (2009), Abstract;
Malarial hemozoin activates the NLRP3 inflammasome through Lyn and Syk kinases: M.T. Shio, et al.; PLoS Pathog. 5, e1000559 (2009), Abstract;
Gout-associated uric acid crystals activate the NALP3 inflammasome: F. Martinon, et al.; Nature 440, 237 (2006), Abstract;

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