KC-6476

293T-cyno-RANKL-Low Cell Line

62870

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Background of 293T-cyno-RANKL-Low Cell Line

RANKL(Receptor Activator of NF-κB Ligand) also known as TNFSF11, TNFSF11 (TNF Superfamily Member 11) is a Protein Coding gene. RANKL is a key molecule that bridges the bone and immune systems. RANKL stimulation activates a signaling pathway downstream of RANK, thereby determining the extent of bone resorption by inducing osteoclast maturation. This protein was shown to activate antiapoptotic kinase AKT/PKB through a signaling complex involving SRC kinase and tumor necrosis factor receptor-associated factor (TRAF) 6, which indicated this protein may have a role in the regulation of cell apoptosis. Diseases associated with TNFSF11 include Osteopetrosis, Autosomal Recessive 2 and Autosomal Recessive Osteopetrosis.

Specifications

Catalog Number	KC-6476
Cell Line Name	293T-cyno-RANKL-Low Cell Line
NCBI/UniProt Accession Number	A0A7N9DBU4
Clone Number	3#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous cyno RANKL gene in low level
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 1:4-1:5 every 2-3 days
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 30 hours
Mycoplasma Status	Negative

Cell Line Generation

293T-cyno-RANKL-Low 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-Low stable clone using FACS.

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

Cell Resuscitation

Pre-warm complete culture medium (DMEM+10% FBS +1μg/mL Puromycin) 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 1:4-1:5 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. Honma M, Ikebuchi Y, Suzuki H. RANKL as a key figure in bridging between the bone and immune system: Its physiological functions and potential as a pharmacological target. Pharmacol Ther. 2021 Feb;218:107682. doi: 10.1016/j.pharmthera.2020.107682. Epub 2020 Sep 19. PMID: 32956720.
2. Raje NS, Bhatta S, Terpos E. Role of the RANK/RANKL Pathway in Multiple Myeloma. Clin Cancer Res. 2019 Jan 1;25(1):12-20. doi: 10.1158/1078-0432.CCR-18-1537. Epub 2018 Aug 9. PMID: 30093448.
3. Nagy V, Penninger JM. The RANKL-RANK Story. Gerontology. 2015;61(6):534-42. doi: 10.1159/000371845. Epub 2015 Feb 14. PMID: 25720990.