KC-0289

293T-cyno-OX40-Cell-Line

20171

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Background of 293T-cyno-OX40-Cell-Line

OX40, also named as CD134 or tumor necrosis factor receptor superfamily, member 4 (TNFRSF4), is a member of the TNFR superfamily acting as co-stimulatory immune checkpoint molecule expressed on the activated T cells. OX40 plays a critical role in maintaining of immune response and lead to a memory immune response. OX40-OX40 Ligand interaction is involved in allergic airway inflammation, graft-versus-host disease and autoimmune disease and cardiovascular disease.

Specifications

Catalog Number	KC-0289
Cell Line Name	293T-cyno-OX40-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	HEK293T cell line stable expressing exogenous cynomolgus 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% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10%FBS+1µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 every 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 OX40 cell line was generated using lentiviral vector expressing cynomolgus OX40 sequence.

Characterization

Figure: Characterization of cyno OX40 overexpressing in 293T stable clones using OX40 antibody.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 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 1:3-1:6 every 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

Ishii, Naoto, Takeshi Takahashi, Pejman Soroosh, and Kazuo Sugamura. 2010. ¡ùOX40Ã¿OX40 Ligand Interaction in T-Cell-Mediated Immunity and Immunopathology.¡ì In, 105:63Ã¿98. Advances in Immunology. Elsevier. doi:10.1016/S0065-2776(10)05003-0.
Aspeslagh, Sandrine, Sophie Postel-Vinay, Sylvie Rusakiewicz, Jean-Charles Soria, Laurence Zitvogel, and Aur¹Èlien Marabelle. 2016. ¡ùRationale for Anti-OX40 Cancer Immunotherapy.¡ì European Journal of Cancer 52 (January). Elsevier: 50Ã¿66. doi:10.1016/j.ejca.2015.08.021.