KC-3379

293T-NFκB-Luc2-TNFRSF11A Cell Line

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Background of 293T-NFκB-Luc2-TNFRSF11A Cell Line

Four heterozygous in-frame tandem duplications of different lengths in TNFRSF11A, the gene that encodes receptor activator of nuclear factor κB (RANK), constitutively activate RANK and lead to high turnover skeletal disease.In humans, monogenic disruption of RANKL/OPG/RANK/NF-κB signaling leads to several extremely rare skeletal disorders that illustrate the importance of this regulatory mechanism [6–11]. Among them, four autosomal dominant seemingly distinctive disorders result from heterozygous in-frame tandem duplications (12-, 15-, 18-, or 27-bp) within exon 1 of TNFRSF11A that encodes the signal peptide of RANK.

Specifications

Catalog Number	KC-3379
Cell Line Name	293T-NFκB-Luc2-TNFRSF11A Cell Line
Host Cell Line	293T-NFκB-Luc2
Description	Stable 293T-NFκB-Luc2 cell line expressing exogenous TNFRSF11A 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+150µg/ml Hygromycin B
Selection Marker	Puromycin, Hygromycin B
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL
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-NFkb-Luc2-TNFRSF11A cell line was generated using lentiviral vector expressing the human TNFRSF11A sequence.

Characterization

Figure 1. Co-culture 293T-NFkb-Luc2-TNFRSF11A cells and 293T-RANKL cells for 6h, then readout with Bright-lite™ Luciferase Assay system.

Figure 2. Co-culture 293T-NFkb-Luc2-TNFRSF11A cells and CHOK1-RANKL cells for 6h, then readout with Bright-lite™ Luciferase Assay system.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS, 150μg/ml Hygromycin and 1µg/mL Puromycin)in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage.

References

1.Iwamoto SJ, Rothman MS, Duan S, Baker JC, Mumm S, Whyte MP. Early-onset Paget's disease of bone in a Mexican family caused by a novel tandem duplication (77dup27) in TNFRSF11A that encodes RANK. Bone. 2020 Apr;133:115224. doi: 10.1016/j.bone.2020.115224. Epub 2020 Jan 8. PMID: 31923705; PMCID: PMC7179970.