RANKL Reporter HEK 293 Cells

RANKL Reporter Cells

HEK-Blue™ RANKL Cells signaling pathway

HEK-Blue™ RANKL cells were engineered from the human embryonic kidney HEK293 cell line to detect bioactive human and murine RANKL by monitoring the activation of the NF-κB and AP-1 pathways. In addition, these cells can be used for screening anti-RANKL antibodies, or inhibitors targeting the RANKL/RANK signaling pathway. 
Receptor Activator of NF-κB Ligand (RANKL) exists as a soluble or transmembrane protein produced by osteoblasts and activated T cells [1]. 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 [1]. 

 More details

Cell line description:

HEK-Blue™ RANKL cells were generated by stable transfection of human embryonic kidney HEK 293 cell line with the human gene for RANK (with all three functional TRAF-binding motifs) and an NF-κB/AP1-inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene.
The binding of RANKL to its receptor on the surface of HEK-Blue™ RANKL cells triggers a signaling cascade leading to the activation of NF-κB/AP1, and the subsequent production of SEAP.  For your convenience, InvivoGen provides QUANTI‑Blue™ Solution, an easy and rapid means to detect and quantify SEAP activity in the cell culture supernatant.  

Features of HEK-Blue™ RANKL cells:

• Fully functional RANKL signaling pathway
• Readily assessable SEAP reporter activity
• Functionally tested
• Guaranteed mycoplasma-free

Applications of HEK-Blue™ RANKL cells:

• Detection of both human and mouse RANKL
• Screening of anti-RANKL antibodies
• Screening of inhibitors targeting RANKL/RANK signaling

References:

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.

Figures

Dose-response of HEK-Blue™ RANKL cells to recombinant RANKL.

Cells were stimulated with increasing concentrations of recombinant human or murine 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 630 nm. EC50 values are shown as mean ± SD.

Cytokine response of HEK-Blue™ RANKL cells.

Cells were stimulated with various human and murine recombinant cytokines: 10 ng/ml of hRANKL, mRANKL, hIL‑1β, mIL‑1β, 1x104 IU/ml of hIFN-α, hIFN-β, 100 ng/ml of hIFN-γ, hTNF-α, and 1 µg/ml of the TLR3 ligand Poly(I:C) HMW and the TLR5 ligand FLA-ST (flagellin from S. typhimurium). After overnight incubation, SEAP activity was assessed using QUANTI‑Blue™ Solution. 

Dose-dependent inhibition of HEK‑Blue™ RANKL cellular response using the Anti-hRANKL-hIgG2 antibody.

Increasing concentration of InvivoGen's Anti-hRANKL-hIgG2 antibody was incubated with HEK‑Blue™ RANKL cells for 30 minutes before the addition of 10 ng/ml of hRANKL. After overnight incubation, SEAP activity in the cell culture supernatant was assessed using QUANTI‑Blue™ Solution. Data represent % inhibition of reporter activity without anti-hRANKL antibody.

Specifications

Antibiotic resistance: Blasticidin,  Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2-4 mM L-glutamine, 10% (v/v) heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 100 μg/ml Normocin™

Guaranteed mycoplasma-free

Specificity: human and mouse RANKL

Detection range:

• 3 - 100 ng/ml for human RANKL
• 1 - 100 ng/ml for murine RANKL

These cells are covered by a Limited Use License (See Terms and Conditions).

Contents

• 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)

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

Details

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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