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THP1-Dual™ KI-hSTING-A162 Cells

THP1-Dual™ KI-hSTING-A162 Cells Unit size Cat. code Docs Qty Price
STING (A162 isoform) knockin NF-kB-SEAP and IRF-Lucia Reporter Monocytes
3-7 x 10e6 cells
thpd-a162
+-
$1,510.00

June, 18th 2018 - CONTENTS UPDATE NOTIFICATION
Please note that, for your convenience, these cells are now provided with QUANTI-Blue™ Solution.

STING (A162 isoform) knockin NF-kB-SEAP and IRF-Lucia Reporter Monocytes

THP1-DualKI-STING cells are a family of reporter cells designed to study variants of STING (stimulator of interferon genes).

THP1-DualKI-hSTING-A162 cells were generated from THP1-Dual™ KO-STING cells by knockin of the intronless coding sequence (from the ATG to the TGA) of the A162 hSTING (S162A) variant. The allele A162 contains a unique point mutation (S162A) placed at the cyclic‑dinucleotide-binding site which confers sensitivity to DMXAA, a potent tumor vascular disrupting agent in mice [2]. In the absence of this mutation, DMXAA has no effect on human STING [3].

THP1-Dual™ KI-hSTING-A162 cells are resistant to blasticidin and Zeocin™.

 

1. Yi G. et al., 2013. Single nucleotide polymorphisms of human STING can affect innate immune response to cyclic dinucleotides. PLoS One. 8:e77846.
2. Gao P. et al., 2013. Structure-function analysis of STING activation by c[G(2’,5’)pA(3’,5’)p] and targeting by antiviral DMXAA. Cell 154(4):748-62.
3. Conlon J. et al., 2013. Mouse, but not human STING, binds and signals in response to the vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid. J Immunol 190(10):5216-25.


THP1-Dual™ KI-hSTING-A162 cells were stimulated with hIFN-β (1 x 104 U/ml), VACV-70 /LyoVec™ (1 μg/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 a 24h incubation, 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%).


THP1-Dual™ KI-hSTING-A162 cells were stimulated with hIFN-β (1 x 104 U/ml), VACV-70 /LyoVec™ (1 μg/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 a 24h incubation, NF-κB activation was determined using QUANTI-Blue™, a SEAP detection reagent, and by reading the optical density (OD) at 655 nm.

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Specifications

Antibiotic resistance: Zeocin™blasticidin

Growth medium:  RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 100 μg/ml Normocin™, Pen-Strep (100 U/ml-100 μg/ml)

Quality control

  • The biallelic knockin (KI) of the human STING variant (A162) has been verified by functional assays, PCR and sequencing.
  • Reporter activity has been validated by stimulating the cells with STING ligands, such as c-di-AMP, cGAMP and DMXAA.
  • The cell line stability for 20 passages following thawing has been verified.
  • The cell line is 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-hSTING-A162 cells (3-7 x 106 cells) in freezing medium
  • 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™ (Lucia luciferase detection medium)
  • 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)

IMPORTANT: Cells are shipped frozen. If cells are not frozen upon arrival, contact InvivoGen immediately.

Shipped on dry ice

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Background

STING (stimulator of interferon genes) is essential for the interferon (IFN) response to cytoplasmic foreign or self-DNA. It directly senses cyclic dinucleotides (CDNs), which are important messengers in bacteria and innate immune agonists in mammals [1]. Distinct variants of human STING (hSTING) that affect CDN recognition and signal transduction have been identified: 

• R232 (R71‑G230-R232-R293): the most prevalent in the human population (~60%). Referred as the “wild-type” or 232R-RGR allele [2].

• HAQ (H71A230-R232-Q293): contains three non-synonymous single nucleotide substitutions; R71H, G230A and R293Q. This allele, found in ~20% of the population, is less sensitive to CDNs than the “wild-type” allele [2].

• H232 (R71‑G230-H232-R293): the most commonly used hSTING variant in structural studies [2].

 S154: a gain-of-function mutation resulting in constitutive STING activation [3].

• M155: a gain-of-function mutation resulting in constitutive STING activation [3,4].

A162: a synthetic mutation (S162A) that confers hSTING sensitivity to DMXAA, a tumor vascular disrupting agent in mice [5].

THP1-Dual™ KI-STING cells are a family of reporter cells allowing the study of STING variation by monitoring the activation of the transcription factors ISRE (IFN-stimulated response elements) and NF-kB. They were generated from THP1-Dual™ KO-STING cells, which derive from the human THP-1 monocytes, by stable biallelic knockout of the endogenous HAQ hSTING gene and stable integration of two inducible secreted reporter genes: Lucia luciferase and SEAP (secreted embryonic alkaline phosphatase). The Lucia luciferase gene is under the control of an ISG54 (interferon-stimulated gene) minimal promoter in conjunction with five ISRE. The SEAP gene is driven by an IFN-β minimal promoter fused to five copies of the NF-kB response element. As a result, they allow the simultaneous study of the IFN regulatory factor (IRF) and NF-kB pathways. Both reporter proteins are readily measurable in the cell culture supernatant when using QUANTI-Luc, a Lucia™ detection reagent, and QUANTI-Blue, a SEAP detection reagent.

 

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. Yi G. et al., 2013. Single nucleotide polymorphisms of human STING can affect innate immune response to cyclic dinucleotides. PLOS One. 8:e77846.
3. Liu Y. et al., 2014. Activated STING in a vascular and pulmonary syndrome. N Engl J Med. 371:507-18.
4. Jeremiah N. et al., 2013. Inherited STING-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations. J Clin Invest. 124:5516-20.
5. Gao P. et al., 2014. Binding-pocket and lid-region substitutions render human STING sensitive to the species-specific drug DMXAA. Cell Reports. 8:1668-76.

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