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THP1-Dual™ KI-mSTING Cells

THP1-Dual™ KI-mSTING Cells Unit size Cat. code Docs Qty Price
Murine STING knockin NF-kB-SEAP and IRF-Lucia Luciferase Reporter Monocytes
3-7 x 10e6 cells
thpd-mstg
+-
$1,563.00

NEW PRODUCT

Murine STING knockin dual reporter monocytes

STING signaling in THP1-Dual™ KI-mSTING cells
STING signaling in THP1-Dual™ KI-mSTING cells

THP1-Dual™ KI-mSTING cells stably express the wild-type murine STING (stimulator of interferon genes, mSTING; also known as TMEM173) gene. They were generated by knockin (KI) of the mSTING intronless coding sequence (from ATG to TGA) into THP1-Dual™ KO-STING cells, which derive from the human THP-1 monocytes.

STING is a cytosolic pattern recognition receptor (PRR) that acts as a direct sensor of cyclic dinucleotides (CDNs) [1]. The binding of CDNs to STING leads to TANK-binding-kinase-I (TBK1)-mediated interferon regulatory factor (IRF3) activation and type I interferon (IFN) production [1] and to NF-κB-dependent inflammatory cytokine production [2].

THP1-Dual™ KI-mSTING 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.

As expected, THP1-Dual™ KI-mSTING cells exhibit a robust response to STING agonists such as 2’3’-cGAMP and 3’3’-cGAMP. In addition, these cells respond to DMXAA (5,6-Dimethylxanthenone 4-acetic acid), a potent activator of murine STING-dependent IFN responses, that does not activate human STING [3]. These cells are resistant to Blasticidin and Zeocin™.

 

Features of THP1-Dual™ KI-mSTING Cells:

  • Knockin of the mSTING gene
  • No functional hSTING expression
  • IRF activation readily assessable using the Lucia luciferase reporter gene
  • NF-κB activation readily assessable using the SEAP reporter gene

Validation of THP1-Dual™ KI-mSTING Cells:

  • Murine STING knockin verified by PCR, DNA sequencing, and Western blot
  • Functionally tested

 

References:

 1. Sun L. et al., 2013. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science. 339:786-91.
2. Ishikawa H. & Barber G.N. 2008. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455:674-8.
3. Zhang H. et al., 2015. Rat and human STINGs profile similarly towards anticancer/antiviral compounds. Sci Rep. 5:18035. 

Figures

Validation of mSTING knockin by PCR
Validation of mSTING knockin by PCR

Amplification of the wild‑type STING gene in THP1-Dual™ cells (WT), which endogenously express full-length hSTING (~5300 bp), and THP1-Dual™  KI-mSTING cells (KI) which feature the full-length intronless mSTING gene (~1000 bp; bottom arrow) and the hSTING gene with a 66-base pair deletion (~5234 bp; top arrow).

Validation of mSTING knockin by Western blot (WES by Protein Simple)
Validation of mSTING knockin by Western blot (WES by Protein Simple)

Lysates from THP1-Dual™ (WT-1), THP1-Dual™  KI-mSTING (KI) and RAW-Dual™  (WT-2; which endogenously express wild-type mSTING) cells were analyzed using Anti-hSTING (1:50 dilution) and Anti-h/mSTING (1:50 dilution). This was followed by HRP-conjugated anti-mouse secondary antibody (undiluted). The arrow indicates the expected size for the human and murine STING protein (40 kDa).  

IRF activation (Lucia luciferase reporter)
IRF activation (Lucia luciferase reporter)

THP1-Dual™ KI-mSTING cells were stimulated with human IFN-β (1 x 104 U/ml), DMXAA (100 µg/ml), 2’3’-cGAMP (30 µg/ml), 3’3’-cGAMP (30 µg/ml), 2’3’-cGAM(PS)2 (Rp/SP) (30 μg/ml), 3’3’-cGAMP fluorinated (30 µg/ml), 2’3’-c-di-AMP (30 µg/ml), 3’3’-c-di-AMP (30 µg/ml), 2’3’-c-di-AM(PS)2 (Rp/SP) (30 µg/ml), 2’3’-c-di-GMP (30 µg/ml) and 3’3’-c-di-GMP (30 µg/ml). A) After 24 hours, IRF activation was determined by measuring the relative light units (RLUs) in a luminometer using QUANTI-Luc™, a Lucia luciferase detection reagent. The IRF induction of each ligand is expressed relative to that of hIFN-β at 1x104 U/ml (taken as 100%).

NF-kB activation (SEAP reporter)
NF-kB activation (SEAP reporter)

THP1-Dual™ KI-mSTING cells were stimulated with human IFN-β (1 x 104 U/ml), DMXAA (100 µg/ml), 2’3’-cGAMP (30 µg/ml), 3’3’-cGAMP (30 µg/ml), 2’3’-cGAM(PS)2 (Rp/SP) (30 μg/ml), 3’3’-cGAMP fluorinated (30 µg/ml), 2’3’-c-di-AMP (30 µg/ml), 3’3’-c-di-AMP (30 µg/ml), 2’3’-c-di-AM(PS)2 (Rp/SP) (30 µg/ml), 2’3’-c-di-GMP (30 µg/ml) and 3’3’-c-di-GMP (30 µg/ml). After 24 hours, NF-κB activation was determined using QUANTI-Blue™ Solution,  a SEAP detection reagent, and by reading the optical density (OD) at 655 nm. 

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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:

  • Murine STING knockin 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

  • 1 vial of THP1-Dual™ KI-mSTING cells (3-7 x 106 cells) in freezing medium
  • 1 ml of Blasticidin (10 mg/ml)
  • 1 ml of Zeocin™ (100 mg/ml)
  • 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.
  • 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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Details

STING (stimulator of interferon genes; also known as TMEM173, MITA, MPYS, and ERIS) is essential for the interferon (IFN) response to microbial or self-DNA, and acts as a direct sensor of cyclic dinucleotides (CDNs). CDNs are important messengers in bacteria and innate immune agonists in mammals.
 Upon bacterial infection, CDNs released into cells bind directly to STING leading to TANK-binding-kinase-I (TBK1)-mediated IFN regulatory factor (IRF3) activation and type I IFN production. Distinct variants of human STING (hSTING) that affect CDN recognition and signal transduction have been identified.

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