RANKL Reporter HEK 293 Cells

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RANKL-responsive NF-κB-SEAP reporter cells

Signaling pathways in HEK-Blue™ RANKL 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).

Figures

Dose-response of HEK-Blue™ RANKL cells to recombinant RANKL. Cells were stimulated with increasing concentrations of recombinant human or mouse RANKL. After overnight incubation, the NF-κB response was determined using QUANTI‑Blue™ Solution, a SEAP detection reagent, and reading the optical density (OD) at 650 nm.

HEK-Blue™ RANKL cell responses to cytokines. Cells were stimulated with 10 ng/ml of hRANKL, mRANKL, 1 ng/ml of hTNF-α, or 100 ng/ml of hTL1A and hCD40L. After overnight incubation, SEAP activity was assessed using QUANTI‑Blue™ Solution. The optical density (OD) at 650 nm is shown as mean ± SEM.

Dose-dependent inhibition of HEK-Blue™ RANKL cells response using Denosumab biosimilar. A serial dilution of Denosumab, a biosimilar Anti-hRANKL-hIgG2 monoclonal antibody (mAb) was incubated with 3 ng/ml of recombinant human RANKL for 2 hours prior to the addition of the HEK-Blue™ RANKL cells. After overnight incubation, the NF-kB response was determined using QUANTI-Blue™ Solution, a SEAP detection reagent. Data are presented as percentage of activity.

Specifications

Cell type: Epithelial

Tissue origin: Human Embryonic Kidney

Target: RANKL

Specificity: Human, mouse

Reporter gene: SEAP

Antibiotic resistance: Blasticidin, Zeocin®

Detection ranges: 

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

Growth medium: Complete DMEM (see TDS)

Growth properties: Adherent

Mycoplasma-free: Verified using Plasmotest™

Quality control: Each lot is functionally tested and validated.

Contents

HEK-Blue™ RANKL Cells (hkb-ranklv2)

• 1 vial containing 3-7 x 10⁶ cells
• 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)

HEK-Blue™ RANKL vial (hkb-ranklv2-av)

• 1 vial containing 3-7 x 10⁶ cells

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

Notification:  Reference #hkb-rankl-av can only be ordered together with reference #hkb-rankl.

Details

Cell line description

HEK-Blue™ RANKL cells were generated by stable transfection 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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