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THP1-Dual™ KI-hSTING-R232 Cells

THP1-Dual™ KI-hSTING-R232 Cells Unit size Cat. code Docs Qty Price
STING (R232 isoform) knockin NF-kB-SEAP and IRF-Lucia Reporter Monocytes
3-7 x 10e6 cells
thpd-r232
+-
$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 (R232 isoform) knockin NF-κB-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-R232 cells were generated from THP1-Dual™ KO-STING cells by knockin of the intronless coding sequence (from the ATG to the TGA) of the R232 hSTING variant. Genomic studies indicate that this variant, which contains an arginine at position 232 (R232), is the most prevalent variant with an occurrence (homozygous allele) of ~45‑58% in the human population [2,3]. This isoform is preferentially activated by 2’5’linkage-containing cGAMP isomers [4].

THP1-Dual™ KI-hSTING-R232 cells are resistant to blasticidin and Zeocin™.

 

1. 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.
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. Jin L. et al., 2011. Identification and characterization of a loss-of-function human MPYS variant. Genes Immun. 12:263-9.
4. Zhang X. et al., 2013. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol. Cell. 51:226–235.


THP1-Dual™ KI-hSTING-R232 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-R232 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 (R232) 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.
  • 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-R232 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)

Shipped on dry ice

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

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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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