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Aluminium hydroxide; NLRP3 inflammasome inducer
NLRP3 Inflammasome Inducer
InvivoGen provides Alum Hydroxide, a ready‑to‑use suspension of aluminium hydroxide, designed for in vitro assays. Its ability to induce the NLRP3 inflammasome has been validated using THP1-Null cells. Of note, Alum Hydroxide induces significantly higher levels of IL-1β by THP1-Null cells than Alum Crystals, using the HEK-Blue™ IL-1β cellular assay.
Aluminium salts (also referred to as alum), such as aluminium hydroxide, aluminium phosphate, or potassium aluminium sulfate, are commonly used vaccine adjuvants, although their mode of action in vivo remains unclear. Comprehensive research in vitro clearly demonstrates that alum induces an NLRP3 inflammasome-dependent induction of interleukin-1β (IL-1β) and IL-18 . Of note, alum has been shown to trigger NLRP3 activation through lysosomal destabilization .
The NLRP3 inflammasome is an intracellular multi-protein complex that plays a central role in innate immunity [3, 4]. It is activated by a two-step process; a first signal (‘priming’) is provided by microbial molecules such as lipopolysaccharide (LPS), while the second signal is provided by a wide array of stimuli including bacterial toxins, endogenous molecules or crystalline substances such as alum. This triggers inflammasome multimerization and caspase-1 activation with the subsequent maturation and secretion of IL-1β and IL-18.
Interestingly, in vivo studies indicate a minimal role for the NLRP3 inflammasome in alum’s adjuvanticity . The reason for this divergence between the in vitro and in vivo action of alum needs to be further investigated.
Features of Alum Hydroxide:
- Inducer of the NLRP3 inflammasome in vitro
- Each lot is functionally tested
1. Li H. et al., 2008. Cutting Edge: Inflammasome activation by Alum and Alum’s adjuvant effect are mediated by NLRP3. J Immunol. 181:17-21.
2. Hornung V. et al., 2008. Silica crystals and aluminium salts activate the NALP3 inflammasome through phagosomal destabilization. Nature Immunol. 9:847-856.
3. Schroder K. & Tschopp J., 2010. The inflammasomes. Cell 140(6):821-32.
4. Franchi L. et al.., 2012. . Sensing and reacting to microbes through the inflammasomes. Nat Immunol 13(4)325-32.
5. Wen Y. & Shi Y., 2016. Alum: an old dog with new tricks. Emerg Microbes Infect.5:e25.
THP1-Null cells, primed with LPS (1 µg/ml for 3h), were stimulated with increasing concentrations of Alum Hydroxide. After overnight incubation, IL-1β secretion was analyzed by adding 50 µl of supernatant from treated THP1-Null cells to HEK-Blue™ IL-1β cells. IL-1β-induced activation of NF-κB was assessed by measuring the levels of SEAP in the supernatant of HEK-Blue™ IL-1β cells using QUANTI-Blue™ Solution, a SEAP detection reagent, and by reading the optical density (OD) at 630 nm.
THP1-Null cells were primed with LPS (1 µg/ml for 3h) and stimulated with increasing concentrations of Alum Hydroxide or Alum Crystals. After overnight incubation, IL‑1β secretion was analyzed by adding 50 µl of supernatant from treated THP1-Null cells to HEK‑Blue™ IL-1β cells. IL-1β‑induced activation of NF-κB was assessed by measuring the levels of SEAP in the supernatant of HEK-Blue™ IL-1β cells using QUANTI-Blue™ Solution, a SEAP detection reagent, and by reading the optical density (OD) at 630 nm.
Working concentrations: 50 - 500 µg/ml
CAS number: 21645-51-2
Chemical formula: Al(OH)3
Molecular weight: 78 g/mol
Form: Liquid (a ready-to-use suspension)
- The biological activity has been validated using cellular assays.
- The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cells.
- 500 µl of Alum Hydroxide (aluminium hydroxide suspension)
This product is provided as a white suspension at 30 mg/ml.
Product is shipped at room temperature
Store at room temperature. DO NOT FREEZE.Back to the top
THP-1/HEK-Blue™ IL-1β Assay
Alum can be used to induce the NLRP3 inflammasome in cellular assays, such as InvivoGen’s THP1/HEK-Blue™ IL-1β assay. This assay uses the secretion of IL-1β by THP1-Null cells as an indicator of NLRP3 inflammasome induction. The production IL-1β by these cells is measured using HEK-Blue™ IL-1β cells.
1- Production of IL-1β by THP-1 cells: Typically, THP1-Null cells are primed with lipopolysaccharide (LPS). These treatments induce the production of pro-IL-1β, the immature form of IL-1β. Subsequent stimulation with inflammasome inducers, such as Alum, leads to NRLP3 and caspase-1 activation resulting in IL-1β maturation and secretion.
2- Detection of IL-1β by HEK-Blue™ IL-1β cells: IL-1β-containing THP-1 supernatants are added to HEK-Blue™ IL-1β cells leading to NF-κB activation and the subsequent production of secreted embryonic alkaline phosphatase (SEAP). The presence of SEAP in HEK-Blue™ IL-1β supernatants is assessed using QUANTI-Blue™ Solution, a SEAP detection medium.Back to the top