KC-1114

293T-cyno-OX40L-Cell-Line

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Background of 293T-cyno-OX40L-Cell-Line

OX40L, also named CD252 and TNSF4, is a type II transmembrane glycoprotein belonging to TNF superfamily, OX40L is the ligand for OX40 (CD134) and mainly expressed on the surface of activated B cells, T cells, DCs and endothelial cells. The interaction of OX40 and OX40L play a important role in late co-stimulatory signal to enhance the survival and proliferation of activated T cells.

Specifications

Catalog Number	KC-1114
Cell Line Name	293T-cyno-OX40L-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous cyno-OX40L gene
Quantity	One vial 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
In Vivo Validation	NA

Cell Line Generation

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

Characterization

Figure 1: Characterization of cyno-OX40L overexpression in the 293T-cyno-OX40L stable clone using FACS.

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.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.
2.Croft M, Esfandiari E, Chong C, Hsu H, Kabashima K, Kricorian G, Warren RB, Wollenberg A, Guttman-Yassky E. OX40 in the Pathogenesis of Atopic Dermatitis-A New Therapeutic Target. Am J Clin Dermatol. 2024 May;25(3):447-461. doi: 10.1007/s40257-023-00838-9. Epub 2024 Jan 18. Erratum in: Am J Clin Dermatol. 2024 May;25(3):463. doi: 10.1007/s40257-024-00850-7. PMID: 38236520; PMCID: PMC11070399.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.