KC-3186

CHOK1-OX40 Cell Line

26915

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Background of CHOK1-OX40 Cell Line

The protein encoded by this gene is a member of the TNF-receptor superfamily. This receptor has been shown to activate NF-kappaB through its interaction with adaptor proteins TRAF2 and TRAF5. Knockout studies in mice suggested that this receptor promotes the expression of apoptosis inhibitors BCL2 and BCL2lL1/BCL2-XL, and thus suppresses apoptosis. The knockout studies also suggested the roles of this receptor in CD4+ T cell response, as well as in T cell-dependent B cell proliferation and differentiation.

Specifications

Catalog Number	KC-3186
Cell Line Name	CHOK1-OX40 Cell Line
Clone Number	25#
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous OX40 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 10μg/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

CHOK1 OX40 cell line was generated using a lentiviral vector expressing the OX40 sequence.

Characterization

Figure 1: Characterization of OX40 overexpression in the CHOK1 OX40 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Lai C, August S, Albibas A, Behar R, Cho SY, Polak ME, Theaker J, MacLeod AS, French RR, Glennie MJ, Al-Shamkhani A, Healy E. OX40+ Regulatory T Cells in Cutaneous Squamous Cell Carcinoma Suppress Effector T-Cell Responses and Associate with Metastatic Potential. Clin Cancer Res. 2016 Aug 15;22(16):4236-48. doi: 10.1158/1078-0432.CCR-15-2614. Epub 2016 Mar 31. PMID: 27034329; PMCID: PMC4987192.
Faghih Z, Abtahi S, Khademi B, Nikfarjam F, Erfani N. Association of OX40 gene polymorphisms (rs17568G/A and rs229811A/C) with head and neck squamous cell carcinoma. Mol Biol Rep. 2019 Jun;46(3):2609-2616. doi: 10.1007/s11033-019-04602-3. Epub 2019 Mar 28. PMID: 30923998.