KC-6316

293T-CTLA4-PD1 Cell Line

61643

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Background of 293T-CTLA4-PD1 Cell Line

PD-1 and CTLA-4 can play an important role in addressing the issue of autoimmune diseases. PD-1 is a transmembrane glycoprotein expressed on T, B, and Dentric cells. This molecule functions as a checkpoint in T cell proliferation. Ligation of PD-1 with its ligands inhibits the production of IL-2, IL-7, IL-10, and IL-12 as well as other cytokines by macrophages, natural killer (NK) cells, and T cells, which can suppress cell proliferation and inflammation. CTLA-4 is a co-receptor on T-cells that controls peripheral tolerance and the development of autoimmunity. Immune check-point blockade (ICB) uses monoclonal antibodies (MAbs) to block the binding of inhibitory receptors (IRs) to their natural ligands.

Specifications

Catalog Number	KC-6316
Cell Line Name	293T-CTLA4-PD1 Cell Line
NCBI/UniProt Accession Number	P16410-1/NM_005018.3
Clone Number	5#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous CTLA4 and PD1 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 + 10μg/mL Blasticidin
Selection Marker	Puromycin \| Blasticidin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 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-PD1 cell line was generated using a lentiviral vector expressing the CTLA4 and PD1 sequence.

Characterization

Figure 1: Characterization of CTLA4 and PD1 overexpression in the 293T-CTLA4-PD1 stable clone using FACS.

Figure 2: Characterization of CTLA4 and PD1 and its mutants overexpressing in 293T stable clones using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 1μg/mL Puromycin + 10μg/mL Blasticidin)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 × 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 1:4-1:5 every 2-3 days; seed out at about 1-3 × 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×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/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. Munir AZ, Gutierrez A, Qin J, Lichtman AH, Moslehi JJ. Immune-checkpoint inhibitor-mediated myocarditis: CTLA4, PD1 and LAG3 in the heart. Nat Rev Cancer. 2024 Aug;24(8):540-553. doi: 10.1038/s41568-024-00715-5. Epub 2024 Jul 9. PMID: 38982146.
2. Kaptein P, Jacoberger-Foissac C, Dimitriadis P, Voabil P, de Bruijn M, Brokamp S, Reijers I, Versluis J, Nallan G, Triscott H, McDonald E, Tay J, Long GV, Blank CU, Thommen DS, Teng MWL. Addition of interleukin-2 overcomes resistance to neoadjuvant CTLA4 and PD1 blockade in ex vivo patient tumors. Sci Transl Med. 2022 Apr 27;14(642):eabj9779. doi: 10.1126/scitranslmed.abj9779. Epub 2022 Apr 27. PMID: 35476594.
3. Brunner-Weinzierl MC, Rudd CE. CTLA-4 and PD-1 Control of T-Cell Motility and Migration: Implications for Tumor Immunotherapy. Front Immunol. 2018 Nov 27;9:2737. doi: 10.3389/fimmu.2018.02737. PMID: 30542345; PMCID: PMC6277866.
4. Rezayi M, Hosseini A. Structure of PD1 and its mechanism in the treatment of autoimmune diseases. Cell Biochem Funct. 2023 Oct;41(7):726-737. doi: 10.1002/cbf.3827. Epub 2023 Jul 20. PMID: 37475518.