ASC expressing RAW 264.7 Cells

ASC expressing RAW 264.7 cells

The role of ASC in canonical 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].

 More details

To foster studies on the ASC adaptor, InvivoGen has developed RAW-ASC cells, which were generated by stable transfection of the murine ASC gene into the murine RAW 264.7 macrophage cell line, which is naturally ASC-deficient [2].

• RAW-ASC cells –  Murine ASC gene expression 

In contrast to the parental cell line, RAW-ASC cells produce and secrete mature IL-1β upon activation of the canonical inflammasome sensors NLRP3 (using Nigericin) and AIM2 (using Poly(dA:dT)), as well as upon activation of the non-canonical inflammasome (using LPS or E. coli outer membrane vesicles). This cell line is a useful tool to study the role of ASC in inflammasome responses and is an alternative to the in vitro differentiation of mouse bone-marrow-derived macrophages. Additionally, it is the parental and control cell line of InvivoGen's  RAW-ASC KO-GSDMD cells, for which all inflammasome responses are abrogated.

FEATURES OF RAW-ASC CELLS:

• Verified expression of the murine ASC gene by Western Blot (Wes™)
• Validated secretion of mature IL-1β and pyroptosis after canonical and non-canonical inflammasome activation
• Parental cell line for RAW-ASC KO-GSDMD cells and RAW-ASC KO-CASP11 cells

Download our Practical guide on Inflammasomes

References:

1. Mathur A. et al., 2017. Molecular mechanisms of inflammasome signaling. J. Leuk. Biol. 103:233.
2. Pelerin P. et al., 2008. P2X7 receptor differentially couples to distinct release pathways for IL-1β in mouse macrophage. J. Immunol. 180:7147.

Figures

Lysates from RAW 264.7 (WT) and RAW-ASC cells were analyzed by Western blot (Wes™) using an Anti-mouse ASC antibody, followed by an HRP-conjugated anti-rabbit secondary antibody. The arrow indicates the expected band for the ASC protein (27 kDa).

~2x10⁵ RAW‑ASC cells were incubated for 3h at 37°C with Pam3CSK4 (100 ng/ml) (priming) and then incubated (activation) with (A) canonical inflammasome inducers Nigericin (5 μM) or transfected Poly (dA:dT) (1 μg/ml), and (B) non-canonical inflammasome inducers, E. coli outer membrane vesicles (OMVs) (20 μg/ml) or transfected LPS-EB (5 μg/ml). For non‑canonical inflammasome activation, cells were pre-primed with recombinant murine IFN-γ (10 ng/ml) overnight at 37°C before priming. After 6h activation, the secretion of mature IL-1β was assessed in the culture supernatant using an ELISA assay.

Specifications

Antibiotic resistance: Blasticidin

Growth medium: DMEM, 4.5 g/l glucose, 4 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml Normocin™

Test medium: DMEM without phenol red, 4.5 g/l glucose, 4 mM L-glutamine, 10% heat-inactivated FBS, 100 U/ml penicillin, 100 µg/ml streptomycin.
Note: Phenol red causes high background signal in the LDH (lactate dehydrogenase) assay used to monitor inflammasome-induced cell death.

Quality Control:

• ASC transfection has been verified by Western blot (WES™) and functional assays.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free. 

This product is covered by a Limited Use License (See Terms and Conditions).

Contents

• 3-7 x 10⁶ RAW-ASC cells in a cryovial or shipping flask
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasma, bacteria, and fungi.
• 1 ml of Blasticidin (10 mg/ml)

 Shipped on dry ice (Europe, USA & Canada)

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 inflammasome sensors that do not contain a CARD domain, such as NLRP3, AIM2, and Pyrin [1]. This is due to the bipartite composition of ASC, consisting of one PYD and one CARD domain, allowing the recruitment of the CARD-containing pro-caspase-1 to these sensors. Whereas, the NLRP1 and NLRC4 inflammasome sensors have a CARD domain, and can recruit pro caspase-1 either directly or through ASC. However, it has been shown that upon activation of these sensors, in the absence of ASC, the secretion of mature IL-1β and IL-18 is reduced [1]. As the non-canonical inflammasome (i.e. CASP4/5/11) can not directly activate CASP1, they trigger GSDMD-driven release of alarmins and K+ efflux to induce the activation of NLRP3 and CASP1-mediated IL-1β/-18 maturation and secretion. Therefore, the absence of ASC affects the downstream inflammatory signaling from the non-canonical inflammasome also. 

1. Mathur A. et al., 2017. Molecular mechanisms of inflammasome signaling. J. Leuk. Biol. 103:233.

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