RAW-ASC cell line細(xì)胞株

ASC (apoptosis-associated speck-like protein containing a CARD domain, also known as PYCARD) is a protein adaptor important in canonical inflammasome responses

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

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.

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.

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