RANKL Reporter HEK 293 Cells
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Human & Mouse RANKL Reporter Cells
3-7 x 10e6 cells
RANKL Reporter Cells
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.
The tumor necrosis factor (TNF)-family member Receptor Activator of NF-κB Ligand (RANKL), also known as member 11 of the TNF superfamily (TNFSF11) or TNF-related activation-induced cytokine (TRANCE), 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 .
RANKL binds to RANK via an obligate trimer configuration [1, 2]. Upon RANKL binding, RANK trimers recruit TNF receptor-associated factor (TRAF) adaptor proteins, such as TRAF6, to TRAF-binding motifs within their cytoplasmic domains . 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].
HEK-Blue™ RANKL cells were generated by stable transfection of HEK293 cells 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. 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
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.
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 an anti-hRANKL antibody.
The antibody was incubated with HEK‑Blue™ RANKL cells for 30 minutes prior to 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.
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™
Specificity: human and mouse RANKL
- 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).Back to the top
- 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)
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