RANKL Reporter HEK 293 Cells

NF-κB-SEAP reporter cells

ABOUT

RANKL-responsive NF-κB-SEAP reporter cells

HEK-Blue™ RANKL cells are designed to monitor human RANKL-induced NF-κB/AP-1 stimulation or inhibition through SEAP detection. This colorimetric bioassay can be used for screening activatory molecules, such as engineered cytokines, or inhibitory molecules, such as neutralizing antibodies.

HEK-Blue™ RANKL cells respond specifically to recombinant human RANKL. The reliable and consistent performance of HEK-Blue™ RANKL cells makes them suitable for release assays of therapeutic molecules that inhibit RANKL signaling, such as Denosumab, a monoclonal antibody blocking the interaction between RANKL and its receptor RANK (see figures).

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Key features

  • Readily assessable NF-κB/AP1-inducible SEAP reporter activity
  • Convenient readout using QUANTI-Blue™ Solution
  • Strong response to human (h) and mouse(m) RANKL
  • Stability guaranteed for 20 passages

Applications

  • Therapeutic development
  • Drug screening
  • Release assay

 

Receptor Activator of NF-κB Ligand (RANKL) is a member of the TNF (Tumor Necrosis Factor) superfamily. This cytokine exists as a soluble or transmembrane protein produced by osteoblasts and activated T cells. RANKL binding to its receptor RANK and the subsequent signaling events play a pivotal role in bone remodeling and dendritic cell survival, thereby enhancing the induction of T cell responses. Blocking the binding of RANKL to its receptor RANK has been shown to reduce osteoporosis, prevent skeletal-related events (SREs) from bone metastasis in cancer, or improve anti-tumor immunity.

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Note: A new clone is provided with an improved RANKL response. The cat code has been changed accordingly (hkb-ranklv2).

Disclaimer:  These cells are for internal research use only and are covered by a Limited Use License (See Terms and Conditions). Additional rights may be available.

SPECIFICATIONS

Specifications

Target

RANKL

Tested applications

Detection of RANKL activity

Cell type
Epithelial
Growth properties
Adherent
Tissue origin
Human embryonic kidney cells
Reporter gene
SEAP
Detection method
Colorimetric
Detection range

Human RANKL: 3 - 100 ng/ml
Mouse RANKL: 1 - 100 ng/ml

Antibiotic resistance
Blasticidin
Zeocin®
Growth medium

Complete DMEM (see TDS)

Mycoplasma-free

Verified using Plasmotest™

Quality control

Each lot is functionally tested and validated.

CONTENTS

Contents

  • Product: 
    HEK-Blue™ RANKL Cells
  • Cat code: 
    hkb-ranklv2
  • Quantity: 
    3-7 x 10^6 cells
Includes:
  • 1 ml of Blasticidin (10 mg/ml)
  • 1 ml of Zeocin® (100 mg/ml)
  • 1 ml of Normocin™ (50 mg/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)

Shipping & Storage

  • Shipping method:  Dry ice
  • Storage:

    • Liquid nitrogen vapor
    Stability: 20 passages

Details

Cell line description 

HEK-Blue™ RANKL cells were generated by the stable transfection  of the human embryonic kidney HEK293 cell line with the gene encoding for human RANK (with all three functional TRAF-binding motifs) and an NF-κB/AP1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The binding of RANKL to its receptor triggers a signaling cascade leading to the activation of NF-κB/AP1, and the subsequent production of SEAP. This can be readily assessed in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent. 

HEK-Blue™ RANKL cells respond to human (h) and mouse (m) RANKL. They can also be used for screening and release assay of molecules that inhibit RANKL signaling, such as denosumab, a monoclonal antibody targeting RANKL. Of note, these cells respond to hTNF-α. They do not respond to hTL1A or hCD40L (see figures).

 

RANKL background

Receptor Activator of NF-κB Ligand (RANKL), also known as member 11 of the tumor necrosis factor (TNF) superfamily (TNFSF11) or TNF-related activation-induced cytokine (TRANCE), exists as a transmembrane or soluble protein produced by osteoblasts and activated T cells [1]. RANKL binds to its receptor RANK via an obligate trimer configuration [1, 2].
RANKL/RANK signaling plays a pivotal role in bone remodeling and dendritic cell survival, thereby enhancing the induction of T cell responses [1]. Upon RANKL binding, RANK trimers recruit TNF receptor-associated factor (TRAF) adaptor proteins, such as TRAF6, to TRAF-binding motifs within their cytoplasmic domains [1].
The TRAF6 signaling cascade results in the activation of NF-κB and AP-1 transcription factors. Multiple efforts have focused on the development of anti‑RANKL antibodies or small‑molecule inhibitors for blocking RANKL/RANK signaling to reduce osteoporosis, prevent skeletal-related events (SREs) from bone metastasis in cancer, or improve anti-tumor immunity [1-3].

 

1. Cheng ML. & Fong L., 2014. Effects of RANKL-targeted therapy in immunity and cancer. Front. Oncol. 3:329.
2. Ahern E. et al., 2018. Roles of the RANKL-RANK axis in anti-tumour immunity — implications for therapy. Nat. Rev. Clin. Oncol. 15:676-93.
3. Nakai Y. et al., 2019. Efficacy of an orally active small-molecule inhibitor of RANKL in bone metastasis. Bone Res. 7:1.

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