KC-1407

293T-cyno-CTLA4 Cell Line

22079

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Background of 293T-cyno-CTLA4 Cell Line

CTLA4 (Cytotoxic T-Lymphocyte-Associated Protein 4) is a critical immune checkpoint receptor expressed primarily on activated T cells. It functions as a negative regulator of T-cell activation, acting as a "brake" on the immune system. CTLA4 binds to the costimulatory molecules CD80/CD86 on antigen-presenting cells with higher affinity than the activating receptor CD28, thereby outcompeting CD28 and transmitting inhibitory signals. This mechanism prevents excessive immune responses and autoimmunity. Therapeutically, blocking CTLA4 (e.g., with Ipilimumab) was the first immune checkpoint inhibitor strategy proven effective in cancer immunotherapy, leading to durable responses in metastatic melanoma and establishing a cornerstone of modern oncology.

Specifications

Catalog Number	KC-1407
Cell Line Name	293T-cyno-CTLA4 Cell Line
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous cyno-ctla4 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 cell line was generated using lentivirus expressing cyno-CTLA4 sequence.

Characterization

Figure 1. Characterization of cyno-CTLA4 over-expression in the 293T-cyno-CTLA4 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 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 1:4-1:8 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

Gogas H, Dafni U, Koon H, Spyropoulou-Vlachou M, Metaxas Y, Buchbinder E, Pectasides E, Tsoutsos D, Polyzos A, Stratigos A, Markopoulos C, Panagiotou P, Fountzilas G, Castana O, Skarlos P, Atkins MB, Kirkwood JM. Evaluation of six CTLA-4 polymorphisms in high-risk melanoma patients receiving adjuvant interferon therapy in the He13A/98 multicenter trial. J Transl Med. 2010 Nov 3;8:108. doi: 10.1186/1479-5876-8-108. PMID: 21044351; PMCID: PMC2988721.