KC-5525

293T-CTLA4 cell line

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Background of 293T-CTLA4 cell line

CTLA4 or CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), also known as CD152 (cluster of differentiation 152), is a protein receptor that functions as an immune checkpoint and downregulates immune responses. CTLA4 is constitutively expressed in regulatory T cells but is upregulated only in conventional T cells after activation - a phenomenon particularly notable in cancer. When it binds to CD80 or CD86 on the surface of antigen-presenting cells, it acts as an \"off\" switch. The CTLA-4 protein is encoded by the Ctla4 gene in mice and the CTLA4 gene in humans.

Specifications

Catalog Number	KC-5525
Cell Line Name	293T-CTLA4 cell line
Host Cell Line	293T
Description	Stable HEK293T clone expressing exogenous CTLA4 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

Cell Line Generation

293T-CTLA4 cell line was generated using lentiviral vector expressing CTLA4 sequence

Characterization

Figure 1: Characterization of CTLA4 overexpression in the 293T-CTLA4 stable clone using FACS.

Figure 2: Characterization of CTLA4 overexpression in the 293T-CTLA4 stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1ug/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 x 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 x 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 x g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3 x106cells/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. Rowshanravan B, Halliday N, Sansom DM. CTLA-4: a moving target in immunotherapy. Blood. 2018 Jan 4;131(1):58-67. doi:10.1182/blood-2017-06-741033. Epub 2017 Nov 8. PMID: 29118008; PMCID:PMC6317697.
2.Genio E, Lecca M, Ciccocioppo R, Errichiello E. CTLA4 Alteration and Neurologic Manifestations: A New Family with Large Phenotypic Variability and Literature Review. Genes (Basel). 2025 Mar 3;16(3):306. doi:10.3390/genes16030306. PMID: 40149457; PMCID: PMC11942126.
3.Gough SC, Walker LS, Sansom DM. CTLA4 gene polymorphism and autoimmunity. Immunol Rev. 2005 Apr;204:102-15. doi: 10.1111/j.0105-2896.2005.00249.x. PMID: 15790353.

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.