THP1-Dual™ KO-MyD88 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^™ 4 Lucia/Gaussia, a Lucia and Gaussia luciferase detection reagent, and QUANTI-Blue^™ Solution, 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.

THP1-Dual™ KO-MyD cells are resistant to the selectable markers blasticidin and Zeocin®.

Key features:

• Verified knockout of the hMyD88 gene
• Functionally validated on a selection of TLR4 and other PRR ligands and cytokines
• Distinct monitoring of NF-κB and IRF activation by assessing the SEAP and Lucia luciferase activities

Applications:

• Defining the role of MyD88 in PRR-induced signaling (e.g. TLR2, TLR4), or related cell signaling pathways
• Highlighting possible overlap between MyD88 and other signaling pathways 

Figures

(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.

Specifications

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)

Antibiotic resistance: Blasticidin and Zeocin®

Quality control:

• Biallelic MyD88 knockout 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).

Contents

• 3-7 x 10⁶ THP1-Dual™ KO-MyD cells in a cryovial or shipping flask
• 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)

Shipped on dry ice (Europe, USA, Canada, and some areas in Asia)

Description

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.

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