ASC-GFP Reporter Monocytes

Real-time monitoring of ASC-dependent inflammasomes 

Activation of THP1-ASC-GFP Cells

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]. ASC's bipartite composition, consisting of one PYD and one CARD domain, allows the recruitment of the CARD-containing pro-caspase-1 to canonical inflammasome sensors that do not contain a CARD domain, such as NLRP3, AIM2, and Pyrin [1].

 More details

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.

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.

Download our Practical guide on Inflammasomes

References:

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.

Figures

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.

Specifications

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 (100 U/ml-100 μg/ml)

Quality control: 

• 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.

Contents

• 3-7 x 10⁶ THP1-ASC-GFP cells  in a cryovial or shipping flask
• 1 ml of Normocin™ (50 mg/ml)
• 1 ml of Zeocin® (100 mg/ml)

Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

Details

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 [1]. 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 [2]. 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 [4].

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.

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