THP1-Dual™ KO-MyD Cells
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Human THP-1 Monocytes - MyD88 knockout NF-kB-SEAP and IRF-Lucia Reporter
3-7 x 10e6 cells
MyD88 knockout dual reporter monocytes
THP1-Dual™ KO-MyD cells were specifically designed to monitor MyD88-mediated signaling.
They were generated from THP1-Dual™cells by stable knockout of the MyD88 gene. They 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 consensus transcriptional response element and three copies of the c-Rel binding site.
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™, a Lucia™ detection reagent and QUANTI-Blue™, a SEAP detection reagent.
THP1-Dual™ KO‑MyD cells are unable to respond to the activation of receptors whose signaling is dependent on MyD88, such as TLR2, TLR4, TLR5, TLR8 and IL-1Rs.
These cells remain responsive to MyD88‑independent receptors, such as NOD1 and TNFR. Activation of the IRF pathway is unaffected by the knockout of the MyD88 gene.
(A) The targeted MyD88 region in THP1-Dual™ (WT) and THP1-Dual™ KO‑MyD (KO) cells was amplified by PCR. THP1-Dual™ KO-MyD cells feature a biallelic deletion (arrow). MWM = molecular weight marker. (B) Lysates from THP1-Dual™ (WT) and THP1‑Dual™ KO-MyD (KO) cells were analyzed by Western blot (Wes™) using an anti-human MyD88 antibody, followed by an HRP conjugated anti-rabbit secondary antibody. The arrow indicates the expected band for the MyD88 protein (33 kDa).
MyD88-dependent and -independent responses in THP1-Dual™ and THP1-Dual™ KO-MyD cells. Cells were incubated with 1 ng/ml Pam3CSK4 (TLR1/2 ligand), 100 ng/ml LPS-EB Ultrapure (TLR4 ligand), 1 µg/ml FLA-ST Ultrapure (TLR5 ligand), 10 µg/ml R848 (TLR7/8 ligand), 1 µg/ml C12-iE-DAP (NOD1 ligand), 1 ng/ml IL-1β, and 1 ng/ml TNF-α. After a 24h incubation, NF-kB activation was determined using QUANTI-Blue™, a SEAP detection reagent, and by reading the optical density (OD) at 655 nm.
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 MyD88 knockout is verified by functional assays, PCR and DNA sequencing.
These cells are guaranteed mycoplasma-free.
This product is covered by a Limited Use License (See Terms and Conditions).Back to the top
- 1 vial of THP1-Dual™ KO-MyD 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™
- 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)Back to the top
THP1 reporter cells are a family of cells derived from the human monocytic THP-1 cell line, which naturally expresses many pathogen recognition receptors (PRRs), including Toll-like receptors.
They respond to ligands for certain TLRs; namely, TLR2, TLR1/2, TLR2/6, TLR4, TLR5 and TLR8. These cells can also be used to study DNA sensing pathways, as they are highly responsive to PRR agonists that trigger interferon (IFN) signaling pathways.
THP1‑Dual™ cells feature two reporter genes that enable the simultaneous study of the NF-κB and IFN signaling pathways.Back to the top