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Human MD2 and CD14 Reporter HEK293 Cells (NF-κB)

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HEK-Blue™ hMD2-CD14 cells

Human MD2 and CD14 expressing HEK293 reporter cells (NF-κB pathway)

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3-7 x 10e6 cells

hkb-hmdcd
+-
$1,457

Notification:  This product is for internal research use only. Additional rights may be available. Please visit InvivoGen’s Terms and Conditions.

NF-κB–SEAP reporter HEK293 cells expressing human MD2 and CD14

Signaling pathways in HEK-Blue™ hMD2-CD14 cells
Signaling pathways in HEK-Blue™ hMD2-CD14 cells

HEK-Blue™ hMD2-CD14 cells were engineered from the human embryonic kidney HEK293 cell line. These cells represent a great tool for studying different Toll-like receptor 4 (TLR4) mutations (gain-of-functions, loss-of-function) since they feature the important adaptor proteins MD2 (myeloid differentiation factor 2) and CD14 (cluster of differentiation 14) without the interference of endogenous TLR4 expression.
 

Description

HEK-Blue™ hMD2-CD14 cells stably express MD2 and CD14, two adaptor proteins crucial for TLR4 signaling. Moreover, these cells harbor an inducible reporter gene for SEAP (secreted embryonic alkaline phosphatase). SEAP levels produced upon TLR4 stimulation can be readily determined by performing the assay in HEK-Blue™ Detection, a cell culture medium that allows for real-time detection of SEAP. Alternatively, SEAP activity may be monitored using QUANTI-Blue™, a SEAP detection reagent.

HEK-Blue™ hMD2-CD14 cells do not express the Toll-like receptor 4 (TLR4). Therefore, they don't respond to any type of LPS, when compared to HEK-Blue™ hTLR4 - a cell line that stably expresses TLR4, MD2, and CD14. In addition, this cell line can be used to distinguish between TLR4- and other TLRs-mediated responses, as HEK293 cells express endogenous levels of various PRRs, including TLR3, TLR5, and RIG-I-like receptors. The parental cell line of HEK-Blue™ hMD2-CD14 cells is HEK-Blue™ Null2 cells.

 

The two adaptor proteins MD2 and CD14 are essential for TLR4-dependent sensing of lipopolysaccharide (LPS). LPS is a major constituent of the outer membrane of Gram-negative bacteria and the main microbial mediator implicated in the pathogenesis of septic shock [1].

More details More details

 

Key features

  • Stable expression of human MD2 and CD14
  • No endogenous expression of human TLR4
  • Distinct monitoring of NF-κB activation by assessing the SEAP activities

Applications

  • Investigating different TLR4 variants (loss-of-function, gain-of-function mutations,...)
  • Defining the role of MD2 and CD14 in LPS-induced signaling pathways 
  • Screening for TLR4 agonists or antagonists
  • Highlighting the importance of TLR4 when used in combination with HEK-Blue™ hTLR4 

 

1. Godowski, P., 2005. A smooth operator for LPS responses. Nat Immunol 6, 544–546.

Figures

Response to TLR4 agonists after transient transfection of hTLR4
Response to TLR4 agonists after transient transfection of hTLR4

Response of HEK-Blue™ hMD2-CD14 cells to TLR4 agonists after transient transfection of hTLR4. Cells were transiently transfected with a hTLR4A plasmid and stimulated with human TNF-α (NF-κB-positive control, 10 ng/ml), LPS-EB UP (1 µg/ml), and LPS-EK UP (1 µg/ml). After overnight incubation, the activation of NF-κB was assessed by measuring the activity of SEAP in the supernatant using QUANTI-Blue™ Solution. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

Response of HEK-Blue™-derived cells to TLR4 agonists
Response of HEK-Blue™-derived cells to TLR4 agonists

Response of HEK-Blue™-derived cells to TLR4 agonists. HEK-Blue™ Null2, HEK-Blue™ hMD2-CD14, and HEK-Blue™ hTLR4 cells were cultured in HEK-Blue™ Detection reagent and stimulated for 24 hours with 10 ng/ml of the following TLR4 agonists: LPS-EB Ultrapure (UP) and LPS-EK UP. Human TNF-α (1 ng/ml) serves as an NF-κB-positive control. After 24h incubation, the NF-κB-induced SEAP activity was assessed by measuring the SEAP level in the supernatant. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

Response of HEK-Blue™ hMD2-CD14 cells to various PRR agonists and cytokines
Response of HEK-Blue™ hMD2-CD14 cells to various PRR agonists and cytokines

Response of HEK-Blue™ hMD2-CD14 cells to various PRR agonists and cytokines. Cells were cultured in HEK-Blue™ Detection reagent and stimulated for 24 hours with cytokines and various PRR agonists: Human TNF-α (NF-κB-positive control, 10 ng/ml), Pam3CSK4 (TLR2 ligand, 1 µg/ml), Poly(I:C) HMW (TLR3 ligand, 1 µg/ml), LPS-EK UP (TLR4 ligand, 1 µg/ml), FLA-ST UP (TLR5 ligand, 1 µg/ml), R848 (TLR7/8 ligand, 10 µg/ml), ODN 2006 (TLR9 ligand, 10 µg/ml), Tri-DAP (NOD1 ligand, 10 µg/ml), and MDP (NOD2 ligand, 10 µg/ml). After 24h incubation, the NF-κB-induced SEAP activity was assessed by measuring the SEAP level in the supernatant. Data are shown as OD at 650 nm (mean ± SEM).

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Specifications

Antibiotic resistance: hygromycin, Zeocin®.

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine, 10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™.

Guaranteed mycoplasma-free.

 

This product is covered by a Limited Use License (See Terms and Conditions).

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Contents

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

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Citations

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