KC-5543

293T-mouse-OX40L Cell Line

56826

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Background of 293T-mouse-OX40L Cell Line

TNFRSF4 (TNF Receptor Superfamily Member 4) also known as OX40L, it is a Protein Coding gene. The tumour necrosis factor receptor OX40 (CD134) is activated by its cognate ligand OX40L (CD134L, CD252) and functions as a T cell co-stimulatory molecule. OX40L is found on antigen-presenting cells, activated T cells and others including lymphoid tissue inducer cells, some endothelia and mast cells. Expression of OX40-OX40L both molecules is increased after antigen presentation occurs and also in response to multiple other pro-inflammatory factors including CD28 ligation, CD40L ligation and interferon-gamma signaling. Diseases associated with TNFRSF4 include Immunodeficiency 16 and Hemolytic Uremic Syndrome, Atypical 6.

Specifications

Catalog Number	KC-5543
Cell Line Name	293T-mouse-OX40L Cell Line
Clone Number	6#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous mouse OX40L 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 1:4-1:8 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

Cell Line Generation

293T-mouse OX40L cell line was generated using a lentiviral vector expressing the mouse OX40L sequence.

Characterization

Figure 1: Characterization of mouse OX40L overexpression in the 293T mouse OX40L stable clone using QPCR.

Figure 2: Characterization of mouse OX40L and its mutants overexpressing in 293T stable clones using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/mL puromycin)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

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

References

1. Cheng J, Cen JM, Cai MY, Xu S, Li L, Li ZC, Yang XL, Chen C, Liu X, Xiong XD. Association between TNFSF4 tagSNPs and myocardial infarction in a Chinese Han population. Genet Mol Res. 2015 Jun 11;14(2):6136-45. doi: 10.4238/2015.June.8.11. PMID: 26125814.
2. Webb GJ, Hirschfield GM, Lane PJ. OX40, OX40L and Autoimmunity: a Comprehensive Review. Clin Rev Allergy Immunol. 2016 Jun;50(3):312-32. doi: 10.1007/s12016-015-8498-3. PMID: 26215166.
3. Lu X. OX40 and OX40L Interaction in Cancer. Curr Med Chem. 2021;28(28):5659-5673. doi: 10.2174/0929867328666201229123151. PMID: 33372866.
4. Song R, Zhang H, Liang Z. Research progress in OX40/OX40L in allergic diseases. Int Forum Allergy Rhinol. 2024 Dec;14(12):1921-1928. doi: 10.1002/alr.23469. Epub 2024 Oct 15. PMID: 39404736.