KC-2550

293T-cyno-CTLA4-delta23-Cell-Line

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Background of 293T-cyno-CTLA4-delta23-Cell-Line

CTLA-4 plays a crucial role in the control of T cell activation and tolerance: in general on the one hand, it mediates its immunosuppressive capacity on regulatory T cells (Treg); on the other hand, it expresses CTLA-4 after the activation of resting T cells, which serves as a feedback control mechanism to balance the production of cytokines, such as IFN-γ, T cell differentiation, expansion, cell contact, and migration, by regulating the strength of the adaptive immune response.

Specifications

Catalog Number	KC-2550
Cell Line Name	293T-cyno-CTLA4-delta23-Cell-Line
Host Cell Line	293T
Description	Stable HEK293T clone expressing exogenous cyno-CTLA4-delta23 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
In Vivo Validation	NA

Cell Line Generation

293T-cyno-ctla4-delta23 cell line was generated using a lentiviral vector expressing the cyno-ctla4-delta23 sequence.

Characterization

Figure 1: Characterization of cyno-CTLA4-delta23 overexpression in the 293T cyno-CTLA4-delta23 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 1:4-1:8 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

Sobhani N, Tardiel-Cyril DR, Davtyan A, Generali D, Roudi R, Li Y. CTLA-4 in Regulatory T Cells for Cancer Immunotherapy. Cancers (Basel). 2021 Mar 22;13(6):1440. doi:10.3390/cancers13061440. PMID: 33809974; PMCID: PMC8005092.
Dimeric arrangement of the parathyroid hormone receptor and a structural mechanism for ligand-induced dissociation. PMID: 20172855 PMCID: PMC2852981 DOI: 10.1074/jbc.M109.093138.
Michelson DA, Benoist C, Mathis D. CTLA-4 on thymic epithelial cells complements Aire for T cell central tolerance. Proc Natl Acad Sci U S A. 2022 Nov 29;119(48):e2215474119. doi: 10.1073/pnas.2215474119. Epub
2022 Nov 21. PMID: 36409920; PMCID: PMC9860321.