KC-2027

293T-cyno-TNFSF14 Cell Line

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Background of 293T-cyno-TNFSF14 Cell Line

The protein encoded by this gene is a member of the tumor necrosis factor (TNF) ligand family. This protein is a ligand for TNFRSF14, which is a member of the tumor necrosis factor receptor superfamily, and which is also known as a herpesvirus entry mediator (HVEM). This protein may function as a costimulatory factor for the activation of lymphoid cells and as a deterrent to infection by herpesvirus. This protein has been shown to stimulate the proliferation of T cells, and trigger apoptosis of various tumor cells. This protein is also reported to prevent tumor necrosis factor alpha mediated apoptosis in primary hepatocyte. Two alternatively spliced transcript variant encoding distinct isoforms have been reported.

Specifications

Catalog Number	KC-2027
Cell Line Name	293T-cyno-TNFSF14 Cell Line
Clone Number	1#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous cyno-TNFSF14 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
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-cyno-TNFSF14 cell line was generated using a lentiviral vector expressing the cyno-TNFSF14 sequence.

Characterization

Figure 1: Characterization of cyno-TNFSF14 overexpression in the 293T-cyno-TNFSF14 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS 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

Garrido VT, Proença-Ferreira R, Dominical VM, Traina F, Bezerra MA, de Mello MR, Colella MP, Araújo AS, Saad ST, Costa FF, Conran N. Elevated plasma levels and platelet-associated expression of the pro-thrombotic and pro-inflammatory protein, TNFSF14 (LIGHT), in sickle cell disease. Br J Haematol. 2012 Sep;158(6):788-97. doi: 10.1111/j.1365-2141.2012.09218.x. Epub 2012 Jul 6. PMID: 22775554.
Kim KH, Choi H, Kim HJ, Lee TR. TNFSF14 inhibits melanogenesis via NF-kB signaling in melanocytes. Cytokine. 2018 Oct;110:126-130. doi: 10.1016/j.cyto.2018.04.034. Epub 2018 May 3. PMID: 29730385.