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	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-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.

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.