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TRIF KO Dual Reporter THP1 Cells

THP1-Dual™ KO-TRIF Cells Unit size Cat. code Docs Qty Price
TRIF Knockout - Human THP-1 Reporter Monocytes
3-7 x 10e6 cells
thpd-kotrif
+-
$1,515.00

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TRIF knockout dual reporter monocytes

THP1-Dual™ KO-TRIF cells were generated from the THP1-Dual™ cell line, which is derived from the human THP-1 monocytic cell line, through the stable knockout of the TRIF gene. THP1-Dual™ KO-TRIF cells feature two reporter genes allowing the simultaneous study of the IRF pathway, by monitoring the activity of an inducible secreted Lucia luciferase, and the NF-κB pathway by monitoring the activity of an inducible SEAP (secreted embryonic alkaline phosphatase). Lucia luciferase and SEAP activities are readily assessable in the supernatant using QUANTI-Luc™ and QUANTI-Blue™ Solution detection reagents, respectively.

TIR-domain-containing adapter-inducing interferon-β (TRIF), also known as TIR-containing adaptor molecule-1 (TICAM-1), is a key adaptor molecule in endosomal TLR-dependent signaling cascades. Upon recognition of lipopolysaccharide (LPS) by endocytosed TLR4, TRIF is responsible for mediating activation of interferon regulatory factor 3 (IRF3) and the production of type I interferons (e.g. IFNβ) [1]. Additionally, TRIF has been shown to induce TLR4-dependent 'late' activation of NF-κB. Importantly, the endosomal TLR4-TRIF signaling cascade is independent of the cell surface TLR4-MyD88 signaling pathway [2]. For comparing these two distinct TLR4 signaling cascades, InvivoGen also offers THP1-Dual™ KO-MyD cells.

 

More details
 

NF-κB and IRF signaling pathways in THP1-Dual™ KO-TRIF cells
NF-κB and IRF signaling pathways in THP1-Dual™ KO-TRIF cells

Key Features:

  • Verified knockout of the TRIF gene (PCR, DNA sequencing, and functional assays)
  • Functionally validated with a selection of PRR ligands and cytokines
  • Readily assessable Lucia luciferase and SEAP reporter activities
  • The stability for 20 passages, following thawing, has been verified
  • Guaranteed mycoplasma-free

Applications:

  • Defining the role of TRIF in PRR-induced signaling (i.e. TLR4), or related cell signaling pathways 
  • Highlighting possible overlap between TRIF and other signaling pathways

 

 

Please note: Studying the TRIF signaling pathway with THP1-Dual™-derived cells does not require PMA‑differentiation, however it does significantly increase their sensitivity to IRF-inducers (i.e. TLR4-dependent TRIF activation). Additionally, THP1-Dual™ cells do not respond to TLR3-specific ligands. 

 

References:

1. Ullah, M.O. et al. 2016. TRIF-dependent TLR signaling, its functions in host defense and inflammation, and its potential as a therapeutic target. J Leukoc Biol 100, 27-45.
2. Ciesielska, A. et al. 2020. TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling. Cell Mol Life Sci.

Figures

Validation of TRIF KO. by PCR and Western blot
Validation of TRIF KO. by PCR and Western blot

Validation of TRIF KO. (A) The targeted TRIF region in THP1-Dual™ (WT; blue arrow) parental cells and THP1‑Dual™ KO-TRIF (KO; red arrow) cells was amplified by PCR. THP1-Dual™ KO-TRIF cells feature a frameshift deletion, causing an early stop codon and inactivation of TRIF. (B) Lysates from THP1-Dual™ (WT) and THP1-Dual™ KO-TRIF (KO) cells were analyzed using an anti-human TRIF antibody (green arrow), followed by an HRP‑conjugated anti‑rabbit secondary antibody. As expected a band was detected at ~100 kDa in the WT cells only.

IRF responses in PMA-differentiated THP1-Dual™ KO-TRIF cells
IRF responses in PMA-differentiated THP1-Dual™ KO-TRIF cells

IRF responses in PMA-differentiated THP1-Dual™-derived cells. THP1-Dual™ and THP1-Dual™ KO-TRIF cells were pretreated with PMA (Phorbol 12-myristate 13-acetate; 10 ng/ml for 3 hours). After 3 days recovery, the cells were incubated with 10 ng/ml human (h)TNF-α (NF‑κB‑SEAP positive control), 1 x 104 U/ml hIFN-β (IRF-Lucia positive control), 1 μg/ml VACV70/LyoVec™ (CDS ligand), 1 µg/ml 3p-hpRNA/LyoVec™ (RIG-I agonist), 30 ng/ml LPS-EK Ultrapure (UP; TLR4), 30 ng/ml LPS-SM-UP (TLR4 agonist), 30 ng/ml CRX-527 (TLR4 agonist), 10 ng/ml Pam3CSK4 (TLR2/1 agonist), 10 µg/ml R848 (TLR7/8 agonist), and 30 μg/ml 2’3’-cGAMP (STING agonist). After overnight incubation, the IRF response was assessed by measuring the activity of Lucia luciferase in the supernatant using QUANTI-Luc™. Data are shown as a fold increase over non-induced cells (Lucia luciferase readout).

NF-κB responses in PMA-differentiated THP1-Dual™ KO-TRIF cells
NF-κB responses in PMA-differentiated THP1-Dual™ KO-TRIF cells

NF-κB responses in PMA-differentiated THP1-Dual™-derived cells. THP1-Dual™ and THP1-Dual™ KO-TRIF cells were pretreated with PMA (Phorbol 12-myristate 13-acetate; 10 ng/ml for 3 hours). After 3 days recovery, the cells were incubated with 10 ng/ml human (h)TNF-α (NF‑κB‑SEAP positive control), 1 x 104 U/ml hIFN-β (IRF-Lucia positive control), 1 μg/ml VACV70/LyoVec™ (CDS ligand), 1 µg/ml 3p-hpRNA/LyoVec™ (RIG-I agonist), 30 ng/ml LPS-EK Ultrapure (UP; TLR4), 30 ng/ml LPS-SM-UP (TLR4 agonist), 30 ng/ml CRX-527 (TLR4 agonist), 10 ng/ml Pam3CSK4 (TLR2/1 agonist), 10 µg/ml R848 (TLR7/8 agonist), and 30 μg/ml 2’3’-cGAMP (STING agonist). After overnight incubation, the activation of NF-κB was assessed by measuring the activity of SEAP in the supernatant using QUANTI-Blue™ Solution. Data are shown as optical density (OD) at 630 nm (mean ± SEM).

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Specifications

Antibiotic resistance: Blasticidin and Zeocin™

Growth medium: RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml Normocin™

Quality Control:

  • Biallelic TRIF knockout has been verified by PCR, DNA sequencing, Western blot, 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).

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Contents

  • 3-7 x 106 THP1-Dual™ KO-TRIF 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.
  • 1 ml of Zeocin™ (100 mg/ml)
  • 1 ml of Blasticidin (10 mg/ml)
  • 1 pouch of QUANTI-Luc™
  • 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)

 Shipped on dry ice (Europe, USA & Canada)

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FAQ

Visit our FAQ Any questions about our cell lines ? Visit our frequently asked questions page

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Details

TRIF Background

TIR-domain-containing adapter-inducing interferon-β (TRIF), also known as TIR-containing adaptor molecule-1 (TICAM-1), is a key adaptor molecule in TLR-dependent signaling cascades. Specifically, TRIF is responsible for mediating TLR4-dependent, MyD88 independent signaling upon recognition of lipopolysaccharide (LPS), as well as, TLR3-dependent signaling upon recognition of double-stranded (ds)RNA [1]. Ultimately, TRIF recruitment activates interferon regulatory factor-3 (IRF3) which regulates both type I interferon (IFN) gene expression and a range of IFN-stimulated genes (ISG) [1]. TRIF plays a central role in a range of innate immune responses including anti-viral host defenses, cell death, and homeostasis [1].

Additionally, TRIF has been shown to induce TLR4-dependent late activation of NF-κB through recruitment and activation of TRAF6 or receptor-interacting serine/threonine protein kinases 1 and 3 (RIPK1 and RIPK3) [2,3]. In addition to the key role played by RIPK1 & RIPK3 in necroptosis, their activation in this context has been shown to lead to the acute expression of pro-inflammatory cytokines [2]. Importantly, TLR4-dependent TRIF activation is independent of its activation of the MyD88-dependent (at the cell surface) pathway, which is implicated in ‘early’ activation of NF-κB and a subsequent pro-inflammatory response [3].

 

References:

1. Ullah, M.O. et al. 2016. TRIF-dependent TLR signaling, its functions in host defense and inflammation, and its potential as a therapeutic target. J Leukoc Biol 100, 27-45.
2. Najjar, M. et al. 2016. RIPK1 and RIPK3 Kinases Promote Cell-Death-Independent Inflammation by Toll-like Receptor 4. Immunity 45(1), 46-59.
3. Ciesielska, A. et al. 2020. TLR4 and CD14 trafficking and its influence on LPS-induced pro-inflammatory signaling. Cell Mol Life Sci.

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