Gasdermin D KO RAW-ASC Cells
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GSDMD Knockout & ASC expressing RAW 264.7 cells (murine macrophages)
3-7 x 10e6 cells
Gasdermin D knockout in RAW 264.7 cells
Gasdermin D (GSDMD) is a cytoplasmic protein with a pore-forming ability that has been described as a major actor in both canonical and non-canonical inflammasome responses. To foster studies on GSDMD, InvivoGen has developed RAW-ASC KO-GSDMD cells, which were generated from the RAW-ASC cell line that derives from the naturally ASC deficient RAW 264.7 macrophage cell line . RAW-ASC KO-GSDMD cells stably express the transfected murine ASC gene and have a stable knockout of the gasdermin D (GSDMD) gene.
• RAW-ASC KO-GSDMD cells – Knockout (KO) of the GSDMD gene and expression of the murine ASC gene
In this cell line, both mature IL-1β secretion and pyroptotic cell death are abolished upon canonical and non-canonical inflammasome activation. This cell line is a useful tool to study the role of GSDMD in the inflammasome responses and is an alternative to the in vitro differentiation of mouse bone-marrow-derived macrophages. Additionally, it can be used as a control cell line for the screening of novel therapeutics that target GSDMD.
Gasdermin D background
GSDMD belongs to a family of six and ten gasdermins in humans and mice, respectively, which all have different expression patterns [2,3]. GSDMD consists of two distinct domains, whereby the C-terminal domain exerts an auto-inhibitory function on the N-terminal domain.
GSDMD is cleaved by activated caspase-1 (CASP1) downstream of canonical inflammasome signaling (NLRP1, AIM2, NLRC4, and Pyrin) or CASP4,5 (human), 11 (mouse), the non-canonical inflammasome, upon activation by intracellular LPS. Ultimately, this cleavage allows the release of the GSDMD N-terminal domain, which oligomerizes to form 10-15 nm diameter pores at the cell membrane. These pores allow the release of alarmins (e.g. HMGB1) and the secretion of mature IL-1β and IL-18 inflammatory cytokines. The accumulation of GSDMD pores in the membrane causes cell swelling and rupture, leading to an inflammatory cell death termed pyroptosis [2,3].
Importantly, GSDMD links the canonical and non-canonical inflammasome responses with the pore formation leading to stress signals, such as cytosolic ion concentration imbalances (i.e. K+ efflux) and ATP release, which ultimately induce the activation of NLRP3 and CASP1-mediated IL-1β/-18 maturation and secretion [4, 5].
Features of RAW-ASC KO-GSDMD cells:
- Verified biallelic knockout of the GSDMD gene and stable expression of the ASC gene (Western blot)
- Complete abrogation of mature IL-1β secretion and pyroptosis after canonical and non-canonical inflammasome activation
1. Pelerin P. et al., 2008. P2X7 receptor differentially couples to distinct release pathways for IL-1β in mouse macrophage. J. Immunol. 180:7147.
2. Feng S. et al., 2018. Mechanisms of Gasdermin family members in inflammasome signaling and cell death. J. Mol. Biol. 430:3068.
3. Kovacs S.B. & Miao E.A. 2017. Gasdermins: effectors of pyroptosis. Trends Cell. Biol. 27:673.
4. Groslambert M. & Py B. 2018. Spotlight on the NLRP3 inflammasome pathway. J. Inflamm. Res. 11:359.
5. Mathur A. et al., 2017. Molecular mechanisms of inflammasome signaling. J. Leuk. Biol. 103:233.
Lysates from RAW-ASC (ASC) and RAW-ASC KO-GSDMD (KO) cells were analyzed by Western blot (Wes™) using an Anti-mouse GSDMD antibody, followed by an HRP‑conjugated anti-rabbit secondary antibody. The arrows indicate the expected bands for the full length GSDMD protein (black arrow; 53 kDa) and the cleaved N-terminal domain (grey arrow; 33 kDa).
~2x105 RAW-ASC (WT) parental and RAW-ASC KO‑GSDMD (KO) cells were incubated for 3h at 37°C with Pam3CSK4 (100 ng/ml) (priming) and then incubated (activation) with Nigericin (5 μM) or transfected Poly (dA:dT) (1 μg/ml). After 6h activation, the secretion of mature IL-1β was assessed in the culture supernatant using an ELISA assay.
~2x105 RAW-ASC (WT) parental and RAW-ASC KO‑GSDMD (KO) cells were pre-primed with recombinant murine IFN-γ (10 ng/ml) overnight at 37°C. Following this, the cells were incubated for 3h at 37°C with Pam3CSK4 (100 ng/ml) (priming) and then incubated (activation) with E. coli outer membrane vesicles (OMVs) (20 μg/ml) or transfected LPS-EB (5 μg/ml). After 6h activation, the secretion of mature IL-1β was assessed in the culture supernatant using an ELISA assay.
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.
- Biallelic GSDMD knockout 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.
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- 3-7 x 106 RAW-ASC KO-GSDMD 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)
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