Caspase-4 KO THP-1 Cells
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CASP4 Knockout in THP-1 cells (human monocytes)
3-7 x 10e6 cells
Caspase-4 knockout in THP-1 cells
Human caspase-4 (CASP4) and CASP5, and their murine ortholog CASP11 are a family of inflammatory caspases that play a crucial role in the non-canonical inflammasome response. To foster research on CASP4, InvivoGen has developed THP1-KO-CASP4 cells, which were generated from the widely used human monocytic THP1 cell line through the stable knockout of the caspase-4 (CASP4) gene.
• THP1-KO-CASP4 cells – Knockout (KO) of the CASP4 gene
In this cell line, mature IL-1β secretion is not affected when the canonical inflammasome is activated, however, upon activation of the non-canonical inflammasome mature IL-1β secretion is severely impaired. This cell line is a useful tool to study the role of CASP4 in inflammasome responses and can also be used as a control cell line for the screening of novel therapeutics that target the CASP4 signaling pathways.
CASP4 is a 'non-canonical inflammasome' that senses intracellular lipopolysaccharide (LPS) from Gram-negative bacteria and causes an inflammatory response, independent of TLR4 [1,2]. In the context of bacterial infection, LPS can be released from lysed bacteria as “free” LPS aggregates or actively delivered to the host cell through the release of small spherical bodies that encapsulate many bacterial components (i.e. toxins, DNA, RNA, and LPS) termed outer membrane vesicles (OMVs). Specifically, CASP4 binds to the Lipid A moiety of LPS, which features variable numbers of acylated fatty acid chains depending on the bacterial strain. Interestingly, CASP4 has a broader activity than its murine ortholog with it being able to bind both hexa-acylated (6 chains) as well as under-acylated (4-5 chains) lipid A . Additionally, an IFN-inducible guanylate-binding protein GBP2 is an important co-factor required to trigger CASP4 activation in response to under-acylated LPS only . CASP4 cannot cleave pro-IL-1β/-18, but instead triggers the cleavage of the pore-forming protein gasdermin D (GSDMD), leading to the release of alarmins and K+ efflux from the cell. This ultimately induces the activation of canonical inflammasome response, through NLRP3, and caspase-1-mediated IL-1β/-18 maturation and secretion [1,2].
Features of THP1-KO-CASP4 Cells:
- Verified biallelic knockout of the CASP4 gene (both α and β isoforms) (DNA sequencing, PCR, and Western blot)
- Complete abrogation of mature IL-1β secretion upon non-canonical inflammasome activation only
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.
1. Schmid-Burgk J.L. et al., 2015. Caspase-4 mediates non-canonical activation of the NLRP3 inflammasome in human myeloid cells. Eur. J. Immunol. 45:2911.
2. Baker P.J. et al., 2015. NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5. Eur. J. Immunol. 45:2918.
3. Lagrange B. et al., 2018. Human caspase-4 detects tetra-acylated LPS and cytosolic Francisella and functions differently from murine caspase-11. Nat. Commun. 9:242.
(A) The targeted CASP4 region in THP1 (WT) parental cells and THP1‑KO‑CASP4 (KO) cells was amplified by PCR. THP1-KO-CASP4 cells feature a biallelic deletion (arrow). (B) Lysates from THP1 (WT) parental cells and THP1-KO-CASP4 (KO) cells were analyzed by Western blot (Wes™) using an Anti-human CASP4 antibody, followed by an HRP-conjugated anti-rabbit secondary antibody. The arrow indicates the expected band for the CASP4 protein (48 kDa).
~3x105 THP1 (WT) parental (blue) and THP1‑KO‑CASP4 (red) cells were incubated for 3h at 37°C with LPS-EK (1μg/ml) (priming) and then incubated (activation) with (A) canonical inflammasome inducers, Nigericin (5 μM) or MSU crystals (MSU; 250 μg/ml), and (B) non-canonical inflammasome inducers, E. coli outer membrane vesicles (OMVs) (100 μg/ml) or transfected with LPS-EK (5 μg/ml). For non-canonical inflammasome activation, the cells were pre‑primed with recombinant human 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 HEK-Blue™ IL-1β sensor cells expressing an NF-κB SEAP reporter gene. QUANTI-Blue™ Solution was used to measure SEAP activity. Optical density (OD) was read at 630 nm.
Growth medium: RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml Normocin™
- Biallelic CASP4 knockout has been verified by DNA sequencing, PCR, 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 THP1-KO-CASP4 cells in a cryovial or shipping flask
- 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.
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