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NLRP3 KO & KD THP-1 Cells

THP1-KO-NLRP3 Cells Unit size Cat. code Docs Qty Price
NLRP3 Knockout in THP-1 cells (human monocytes)
3-7 x 10e6 cells
thp-konlrp3
+-
$1,288.00
available soon
THP1-defNLRP3 Cells Unit size Cat. code Docs Qty Price
NLRP3 Knockdown in THP-1 cells (human monocytes)
3-7 x 10e6 cells
thp-dnlp
+-
$1,182.00

NLRP3 knockout or knockdown in THP-1 cells

NLRP3 (NOD-like receptor pyrin domain-containing protein 3, cryopyrin or NALP3) is a cytoplasmic protein and the best-described inflammasome sensor.  To foster research on the NLRP3 sensor, InvivoGen provides cellular tools that have been generated from the human monocytic THP1 cell line through either a stable knockout or knockdown of the NLRP3 gene. THP-1 cells are widely used for inflammasome studies due to their high expression levels of NLRP3, ASC, and pro-caspase-1.

• THP1-KO-NLRP3 cells – Knockout (KO) of the NLRP3 gene

• THP1-defNLRP3 cells – Knockdown (KD) of the NLRP3 gene

 

These cell lines exhibit a similar phenotype but have dramatically different genotypes. They share the common phenotype of abrogation of mature IL-1β secretion. However, THP1- defNLRP3 cells exhibit a knockdown of NLRP3 gene expression whereas THP1-KO-NLRP3 cells possess a KO of the NLRP3 gene. Indeed, THP1- defNLRP3 cells have a maximum 2-fold reduction in NLRP3 expression (RT-qPCR), whereas THP1-KO-NLRP3 cells have no NLRP3 protein expression (Western blot). These cell lines are useful tools in the study of the NLRP3 sensor in inflammasome responses and can also be used as control cell lines for the screening of novel therapeutics that target NLRP3 signaling pathways.

 

Canonical and non-canonical activation of NLRP3
Canonical and non-canonical activation of NLRP3

NLRP3 background

Inflammasomes are cytoplasmic multi-protein complexes that assemble in response to infections and cellular damage. Canonical and non-canonical inflammasomes have been identified. Canonical inflammasomes are characterized by a primary sensor, such as NLRP3, that recruits the ASC adaptor leading to caspase-1 (CASP1) activation.

The canonical inflammasome response requires two signals, priming (recognition of PAMPs or DAMPs by pattern recognition receptors such as TLRs) and activation [1,2]. Activation of NLRP3 can be triggered by a wide range of structurally and chemically unrelated stimuli  (e.g. pore-forming toxins, activators of ion channels, MSU crystals, β-amyloid proteins). Therefore, instead of directly binding to these stimuli, NLRP3 senses downstream cytosolic stress signals such as ion imbalances (e.g. K+ efflux) [2]. This leads to the aggregation of NLRP3 and the ASC adaptor and the cleavage and activation of CASP1. This induces the maturation of pro-IL-1β/pro-IL-18, and cleavage of the pore-forming protein gasdermin D (GSDMD), leading to the secretion of IL-1β/-18 as well as pyroptosis [1,2].

Additionally, NLRP3 is activated indirectly by the induction of the non-canonical inflammasome (CASP4/5 in humans and CASP11 in mice) upon the sensing of cytosolic LPS. These caspases trigger GSDMD‑driven release of alarmins and K+ efflux, which ultimately induces the activation of NLRP3 and CASP1-mediated IL-1β/-18 maturation and secretion [1,2].

 

Features of THP1-KO-NLRP3 cells [NEW]:

  • Verified biallelic KO of the NLRP3 gene (DNA sequencing, PCR, and Western blot)
  • Complete abrogation of mature IL-1β secretion upon activation of NLRP3
  • Differing pyroptotic cell death depending upon activation of NLRP3 by canonical or non-canonical inflammasomes 

Features of THP1-defNLRP3 cells:

  • Verified KD of the NLRP3 gene (RT-qPCR)
  • Significantly reduced mature IL-1β secretion upon activation of the NLRP3 
  • Highly referenced in inflammasome-related literature (see citations)

 

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.

 

Read our review on Inflammasome activation

 

References:

1. Swanson K.V. et al., 2019. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat. Rev. Immunol. 19:477.
2. Groslambert M. & Py B. 2018. Spotlight on the NLRP3 inflammasome pathway. J. Inflamm. Res. 11:359.

Figures

Validation of NLRP3 KO in THP1-KO-NLRP3 cells
Validation of NLRP3 KO in THP1-KO-NLRP3 cells

(A) The targeted NLRP3 region in THP1 (WT) parental cells and THP1-KO-NLRP3 (KO) cells was amplified by PCR. THP1-KO-NLRP3 cells feature a biallelic deletion (arrow). (B) Lysates from THP1 (WT) parental cells and THP1-KO‑NLRP3 (KO) cells were analyzed Western blot (Wes™) using an Anti-human NLRP3 antibody, followed by an HRP-conjugated anti-rabbit secondary antibody. The arrow indicates the expected band for the NLRP3 protein (110 kDa).

Absence of mature IL-1β secretion in KO cells upon inflammasome activation
Absence of mature IL-1β secretion in KO cells upon inflammasome activation

~3x105 THP1 WT parental (blue) and THP1-KO-NLRP3 cells (red) were incubated for 3h at 37°C with LPS-EK (1 μg/ml) (priming) and then incubated (activation) with Nigericin (5 μM), MSU crystals (MSU; 250 μg/ml), transfected Poly (dA:dT) (1 μg/ml), or E. coli outer membrane vesicles (OMVs) (100 μg/ml). For non-canonical inflammasome activation with E. coli OMVs, 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. 

Absence of pyroptosis in KO cells upon inflammasome activation
Absence of pyroptosis in KO cells upon inflammasome activation

~3x105 THP1 WT parental (blue) and THP1-KO-NLRP3 cells (red) were incubated for 3h at 37°C with LPS-EK (1 μg/ml) (priming) and then incubated (activation) with Nigericin ( 0.1-10 μM). After 6h, cell death was assessed using the lactate dehydrogenase (LDH) assay.

Validation of NLRP3 KD in THP1-defNLRP3
Validation of NLRP3 KD in THP1-defNLRP3

Quantitative RT-PCR analysis showing the fold change of NLRP3 and ASC genes in THP1-defNLRP3 cells compared to THP1-null cells.

Reduced mature IL-1β secretion by THP1-defNLRP3 cells
Reduced mature IL-1β secretion by THP1-defNLRP3 cells

Cells were primed with LPS (1 μg/ml) and then induced (activated) with ATP (5 mM) or MSU crystals (MSU; 100 μg/ml). After 24h incubation,  the secretion of mature IL-1β was assessed in the culture supernatants 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.

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Specifications

THP1-KO-NLRP3 cells

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™

Quality Control:

  • Biallelic NLRP3 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. 

 

THP1-defNLRP3 cells

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™

Antibiotic resistance: Hygromycin B

Quality control:

  • NLRP3 deficiency (def) has been verified by qRT-PCR and functional assays.
  • The stability for 20 passages, following thawing, has been verified. 
  • These cells are guaranteed mycoplasma-free. 

 

Both of these products are covered by a Limited Use License (See Terms and Conditions).

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Contents

THP1-KO-NLRP3 cells

  • 3-7 x 106 THP1-KO-NLRP3 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.

Shipped on dry ice (Europe, USA & Canada)

THP1-defNLRP3 cells

  • 3-7 x 106 THP1-defNLRP3 cells in a cryovial or shipping flask
  • 1 ml of Hygromycin B Gold (100 mg/ml)
  • 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.

Shipped on dry ice (Europe, USA & Canada)

 

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Citations

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