ASC-GFP Reporter Monocytes
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Human THP-1 Monocytes - ASC speck reporter cells
3-7 x 10e6 cells
Real-time monitoring of ASC-dependent inflammasomes
ASC (apoptosis-associated speck-like protein containing a CARD domain, also known as PYCARD) is a protein adaptor important in canonical inflammasome responses [1,2]. To foster research on the ASC adaptor, InvivoGen provides cellular tools that have been generated from the human monocytic THP1 cell line. THP-1 cells are widely used for inflammasome studies due to their high expression levels of NLRP3, ASC, and pro-caspase-1.
THP1-ASC-GFP cells are inflammasome reporter cells that allow the monitoring of ASC-dependent inflammasome formation using fluorescence microscopy. These cells stably express a gene encoding an ASC::GFP fusion protein for which expression is driven by an NF-κB-inducible promoter. Hence, in resting cells, no GFP signal is detected. Upon the first step of inflammasome activation (‘priming”), NF-kB-dependent ASC::GFP expression is induced and can be observed throughout the cytoplasm. Following the second step of inflammasome activation, ASC::GFP polymerizes to form a macromolecular, micrometer-sized complex. The presence of ASC::GFP positive cells and localization of fluorescent ASC specks can be determined by using time-lapse confocal or high-resolution fluorescence microscopy.
THP1-ASC-GFP cells are resistant to Zeocin™.
Inflammasomes are multimeric protein complexes that are crucial for host defense against infection and response to endogenous danger signals. The canonical inflammasome response is driven by aggregation of a sensor (i.e. NLRP3) with the ASC adaptor and pro-caspase-1. Activation of caspase-1 (CASP1) induces the maturation of pro-IL-1β/pro-IL-18 and cleavage of the pore-forming protein gasdermin D (GSDMD), leading to secretion of IL-1β/ 18 and pyroptosis.
ASC is essential to canonical inflammasome sensors that do not contain a CARD domain, such as NLRP3, AIM2, and Pyrin [1,2]. ASC's bipartite composition, consisting of one PYD and one CARD domain, allows the recruitment of the CARD-containing pro-caspase-1 to these sensors. The NLRP1 and NLRC4 inflammasome sensors have a CARD domain, and can thus recruit pro-caspase-1 either directly, or through ASC. However, NLRP1 or NLRC4 activation in the absence of ASC triggers a reduced secretion of mature IL-1β and IL-18 . Importantly, non-canonical inflammasomes (i.e. CASP4/5/11) activate CASP1 indirectly: their activation triggers GSDMD-driven release of alarmins and K+ efflux, which in turn, induce NLRP3- and CASP1-mediated IL-1β/-18 maturation and secretion.
In resting cells, ASC is present in a soluble and diffuse form both in the cytoplasm and nucleus . Inflammasome activation in most cells leads to the formation of one large, micrometer-sized, ASC ‘speck’ per cell, thus concentrating CASP1 activation sites [2,3]. Yet, the number of ASC specks may dependent on the inflammasome inducer used. For example, Nigericin promotes the formation of numerous specks, whereas ATP leads to the accumulation of fewer specks .
Features of THP1-ASC-GFP Cells:
- NF-kB-dependent expression of the ASC::GFP fusion protein
- No GFP expression in resting cells
- Intact inflammasome responses
Applications for THP1-ASC-GFP Cells:
- Real-time monitoring of ASC-dependent inflammasome priming and activation steps
- Rapid and visual screening of drugs targeting the ASC signaling by fluorescence microscopy
InvivoGen also provides THP1-KO-ASC cells featuring a biallelic knockout of the human ASC gene. These cells do not express the ASC protein and display a complete abrogation of mature IL-1β secretion upon activation of the canonical and non-canonical inflammasomes.
For detecting and quantifying the release of mature human (h)IL-1β, InvivoGen provides HEK-Blue™ IL-1β sensor cells, which express an NF-κB-inducible SEAP reporter gene. QUANTI-Blue™ Solution allows rapid colorimetric detection and measure of SEAP activity by reading the optical density at 630-650 nm.
For detecting and quantifying pyroptotic cell death, InvivoGen provides THP1-HMGB1-Lucia™ cells, which express a cytoplasmic HMGB1::Lucia luciferase fusion protein that is released in the supernatant upon pyroptosis. The activity of the Lucia luciferase reporter protein can be readily assessed using the QUANTI-Luc™ detection reagent.
1. Mathur A. et al., 2017. Molecular mechanisms of inflammasome signaling. J. Leuk. Biol. 103:233.
2. Hoss F. et al., 2017. Assembly and regulation of ASC specks. Cell. Mol. Life Sci. 74:1211.
3. Stutz A. et al., 2013. ASC speck formation as a readout for inflammasome activation. Methods Mol. Biol.
4. Zha Q. et al., 2016. ATP-induced inflammasome activation and pyroptosis is regulated by AMP-activated protein kinase in macrophages. Front Immunol. 7:597.
THP1-ASC-GFP cells were primed with 1 μg/ml LPS-EK for 3 hours, inducing the expression of the ASC::GFP fusion protein in the cytoplasm (middle panel). Cells were then incubated with 250 ng/ml complexed Poly(dA:dT) and ASC speck formation was monitored over 1 to 3 hours post-activation. In most cells, only one speck forms upon inflammasome activation (arrows in right pannel). Scale bar: 50 μm.
Antibiotic resistance: Zeocin™
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)
- The functionality of THP1 ASC-GFP cells has been tested using inflammasome inducers, such as the microbial toxin nigericin and transfected poly(dA:dT).
- The stability of this cell line for 20 passages following thawing has been verified.
- THP1 ASC-GFP cells are guaranteed mycoplasma-free.
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- 3-7 x 106 THP1-ASC-GFP cells in a cryovial or shipping flask
- 1 ml Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.
- 100 μl Zeocin (100 mg/ml).
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