THP1-Dual™ KO-STING Cells
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Human THP-1 Monocytes - STING knockout NF-κB-SEAP and IRF-Lucia Reporter Cells
3-7 x 10e6 cells
STING knockout dual reporter monocytes
THP1-Dual™ KO-STING cells were generated from THP1-Dual™ cells by stable knockout of the STING gene. STING (stimulator of interferon genes), alternatively known as MPYS, TMEM173, MITA, and ERIS is a direct sensor of cyclic dinucleotides (CDNs) [1-3]. These cell lines were derived from human THP‑1 monocytes, a cell line often used to study DNA sensing pathways as they express all the cytosolic DNA sensors identified so far (with the exception of DAI).
THP1-Dual™ and THP1-Dual™ KO-STING cells stably express two inducible secreted reporter genes: IFN-inducible Lucia luciferase and NF‑κB-inducible SEAP (secreted embryonic alkaline phosphatase). They can be used to study the role of STING by monitoring IRF-induced Lucia luciferase activity, using QUANTI‑Luc™ 4 Lucia/Gaussia, a Lucia and Gaussia luciferase detection reagent.
1. Burdette DL. et al., 2011. STING is a direct innate immune sensor of cyclic di-GMP. Nature. 478(7370):515-8.
2. Zhang X. et al., 2013. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol Cell. 51(2):226-35.
3. Barber GN. et al., 2015. STING: infection, inflammation and cancer. Nat Rev Immunol. 15(12):760-70.
Analysis of lysates (40 µg) using Anti-hSTING-IgG (0.2 µg/ml), followed by HRP-conjugated goat anti-mouse IgG1 secondary antibody (1:3000 dilution).
KO: THP1-Dual™ KO-STING cells. WT: THP1-Dual™ cells (endogenously expresses the HAQ hSTING variant).
IRF responses in THP1-Dual™ and THP1-Dual™ KO-STING cells to various stimuli. Cells were incubated with Poly(I:C) LMW/LyoVec™ (100 ng/ml), VACV-70/LyoVec™ (1 µg/ml), 2'3'-cGAMP (3 µg/ml), and c-di-AMP (10 µg/ml). Human IFN-α and IFN-β (1x104 U/ml) serve as positive controls. 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%).
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)
Biallelic STING knockout is verified by functional assays, PCR and DNA sequencing.
These cells are guaranteed mycoplasma-free.
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- 1 vial of THP1-Dual™ KO-STING 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 tube of QUANTI-Luc™ 4 Reagent, a Lucia luciferase detection reagent (sufficient to prepare 25 ml)
- 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)
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THP1-Dual™ and THP1-Dual™ KO-STING cells stably express two inducible secreted reporter genes: Lucia luciferase and SEAP (secreted embryonic alkaline phosphatase). The Lucia luciferase reporter gene is under the control of an ISG54 (interferon-stimulated gene) minimal promoter in conjunction with five IFN-stimulated response elements. The SEAP gene is driven by an IFN-β minimal promoter fused to five copies of the NF‑κB response element. As a result, they allow the simultaneous study of the IFN regulatory factor (IRF) pathway, by assessing the activity of Lucia luciferase and the NF-κB pathway, by monitoring the activity of SEAP. Both reporter proteins are readily measurable in the cell culture supernatant when using QUANTI-Luc™ 4 Lucia/Gaussia, a Lucia and Gaussia luciferase detection reagent, and QUANTI-Blue™ Solution, a SEAP detection reagent.
THP1-Dual™ KO-STING and THP1-Dual™ cells can be used to study the role of STING by monitoring IRF-induced Lucia luciferase activity. Unlike the parental cells, THP1-Dual™ KO‑STING cells exhibit no detectable response to cytosolic DNA and CDNs while retaining the ability to respond to type I IFNs. As expected these cells remain responsive to RIG-I ligands such as transfected Poly(I:C).
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