KC-6095

293T-cyno-RANKL Cell Line

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Background of 293T-cyno-RANKL Cell Line

RANKL (Receptor Activator of NF-κB Ligand), officially named TNFSF11 (TNF Superfamily Member 11), is also known as OPGL, ODF, and TRANCE . It is a key cytokine for osteoclast differentiation, activation, and survival . Expressed in bone, lymphoid tissues, and activated T-cells, it binds to its receptor RANK to regulate bone remodeling . Dysregulation of the RANKL/RANK/OPG axis is central to diseases like postmenopausal osteoporosis, rheumatoid arthritis, and bone metastases . This has made RANKL a critical therapeutic target, leading to the development of the monoclonal antibody Denosumab, which inhibits its activity . Current research explores engineered RANKL fragments as novel osteoporosis therapies .

Specifications

Catalog Number	KC-6095
Cell Line Name	293T-cyno-RANKL Cell Line
NCBI/UniProt Accession Number	A0A7N9DBU4
Clone Number	1#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous cyno RANKL gene
Quantity	Two vials of frozen cells (≥2-10⁶/vial)
Stability	Stable in culture over a minimum of 10 passages
Application	Drug screening and biological assays
Freezing Medium	70% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial-like
Subculture	Split saturated culture 1:4-1:8 every 2-3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T-cyno-RANKL cell line was generated using a lentiviral vector expressing the cyno RANKL sequence.

Characterization

Figure 1: Characterization of cyno RANKL overexpression in the 293T-cyno-RANKL stable clone using FACS.

Figure 2: Characterization of cyno RANKL in the 293T-cyno-RANKL stable clone using PCR sequencing.

Cell Resuscitation

Pre-warm complete culture medium (basal medium and 10% FBS) in a 37°C water bath.
Rapidly thaw the cryovial in a 37°C water bath for 1-2 minutes with gentle agitation.
Transfer the vial to a biosafety cabinet, and disinfect the exterior with 70% ethanol.
Aseptically transfer the cell suspension dropwise into a sterile centrifuge tube containing 9.0 mL of pre-warmed complete medium.
Centrifuge at approximately 125 × g for 5–7 minutes at room temperature, carefully aspirate the supernatant without disturbing the cell pellet.
Gently resuspend the pellet in an appropriate volume of complete medium and transfer the suspension into a T25 flask.
Incubate the flask in a 37°C in a humidified 5% CO₂ incubator.
Assess cell viability and morphology after 24 hours. If cells appear healthy, replace the medium with fresh medium supplemented with the appropriate selective antibiotic.
Subculture the cells at a ratio of 1:4-1:8 every 2-3 days upon reaching 80%–90% confluency.

Cell Freezing

Prepare the freezing medium (70% basal medium, 20% FBS and 10% DMSO) freshly before use.
Pre-chill the freezing medium on ice and label the cryovials accordingly.
Transfer the cell suspension to a sterile conical tube and perform a cell count to determine total viability and density.
Centrifuge the cells at 250×g for 5 minutes at room temperature; carefully aspirate the supernatant.
Gently resuspend the cell pellet in chilled freezing medium, ensuring a minimum cell density of 3×10⁶ cells/mL.
Aliquot 1 mL of the cell suspension into each pre-labeled cryovial.
Place the cryovials into a CoolCell® container and store at -80°C overnight for controlled-rate cooling.
Transfer the cryovials to the liquid nitrogen for long-term storage the following day.

References

1. Bae, Seyeon et al. “RANKL-responsive epigenetic mechanism reprograms macrophages into bone-resorbing osteoclasts.” Cellular & molecular immunology vol. 20,1 (2023): 94-109. doi:10.1038/s41423-022-00959-x
2. Ando, Yutaro, and Masayuki Tsukasaki. Nihon yakurigaku zasshi. Folia pharmacologica Japonica vol. 158,3 (2023): 263-268. doi:10.1254/fpj.22122
3. Tobeiha, Mohammad et al. “RANKL/RANK/OPG Pathway: A Mechanism Involved in Exercise-Induced Bone Remodeling.” BioMed research international vol. 2020 6910312. 19 Feb. 2020, doi:10.1155/2020/6910312