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Human THP1 monocytes with reduced NLRP3 activity - Inflammasome test cells
3-7 x 10e6 cells
Human monocytes with reduced NLRP3 activity
THP1-defNLRP3 cells are derived from THP-1 human monocytic cells. THP-1 cells are the most commonly used model cell line for the study of inflammasome activation as they express high levels of NLRP3, ASC and pro-caspase-1.
THP1-defNLRP3 cells have reduced NLRP3 activity but are proficient for ASC and caspase-1 activities.
THP1-defNLRP3 cells display significantly weaker responses to inducers of the NLRP3 inflammasome, such as ATP and MSU, when compared to THP1-Null cells. THP1-defNLRP3 cells also show a greatly reduced response to extracellular calcium, a danger signal that activates the NLRP3 inflammasome.
However, THP1-defNLRP3 cells may respond to signals that activate other ASC-dependent inflammasomes, such as NLRP1 and NLRC4 inflammasomes. THP1-defNLRP3 cells are designed as tools to study the involvement of NRLP3 in response to a given signal.
The positive control cell line for inflammasome studies are THP1-Null cells.
Cells primed with LPS (1 μg/ml) were stimulated with ATP (5 mM) or MSU (100 μg/ml). After 24h incubation, the supernatants were added 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 the QUANTI-Blue™ assay.
Quantitative RT-PCR analysis showing the fold change of NLRP3 and ASC genes in THP1-defNLRP3 cells compared to THP1-null cells.
Antibiotic resistance: hygromycin B.
Growth Medium: RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 100 μg/ml Normocin™, Pen-Strep (50 U/ml-50 μg/ml).
- NLRP3 deficiency in THP1-defNLRP3 cells was confirmed by qRT-PCR and a functionality assay using inflammasome inducers.
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THP1-defNLRP3 cells are designed to study the signals involved in inflammasome activation. To become susceptible to inflammasome inducers, these cells must be induced by stimuli commonly used for induction in model systems, such as lipopolysaccharide (LPS) and phorbol 12-myristate acetate (PMA). Stimulation by LPS or differentiation with PMA induces the production of pro-IL-1β, the immature form of IL-1β. Subsequent stimulation with inflammasome inducers, such as ATP and alum crystals, leads to caspase-1 activation and IL-1β maturation and secretion. Mature IL-1β can be detected by Western blot, ELISA, or a cell-based assay.
InvivoGen has developed a new method to detect bioactive IL-1β, based on HEK293 cells specifically engineered to selectively respond to IL-1β, named HEK-Blue™ IL-1β. These cells feature the SEAP (secreted embryonic alkaline phosphotase) reporter gene under the control of an NF-kB-inducible promoter. They naturally express the IL-1β receptor (IL-1R), and all the proteins involved in the MyD88-dependent IL-1R signaling pathway that leads to NF-kB activation. Thus upon IL-1β binding to IL-1R, a signaling cascade is initiated triggering NF-kB activation and the subsequent production of SEAP. Detection of SEAP in the supernatant of HEK-Blue™ IL-1β cells can be readily assessed using QUANTI-Blue™, a SEAP detection medium. QUANTI-Blue™ turns blue in the presence of SEAP which can be easily quantified using a spectrophotometer.Back to the top