KC-1395

293T-rat-CTLA4-Cell-Line

22055

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Background of 293T-rat-CTLA4-Cell-Line

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), also known as CD152, is a leukocyte differentiation antigen, it is an inhibitory receptor belonging to the CD28 immunoglobulin subfamily, expressed primarily by T-cells. CTLA-4 is a critical negative regulator of the immune system and a major target for immunotherapy. Recovery and transient localization of CTLA-4 as a "mobile target" in activated T cell membrane plays an important role in the intrinsic regulation of cell signaling. Diseases associated with CTLA4 include Immune Dysregulation With Autoimmunity, Immunodeficiency, And Lymphoproliferation and Celiac Disease 3.

Specifications

Catalog Number	KC-1395
Cell Line Name	293T-rat-CTLA4-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous rat 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-rat-CTLA4 cell line was generated using a lentiviral vector expressing the rat CTLA4 sequence.

Characterization

Figure 1: Characterization of endogenous rat CTLA4 expression in 293T cells 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

Van Coillie S, Wiernicki B, Xu J. Molecular and Cellular Functions of CTLA-4. Adv Exp Med Biol. 2020;1248:7-32. doi: 10.1007/978-981-15-3266-5_2. PMID: 32185705.
Ovcinnikovs V, Ross EM, Petersone L, Edner NM, Heuts F, Ntavli E, Kogimtzis A, Kennedy A, Wang CJ, Bennett CL, Sansom DM, Walker LSK. CTLA-4-mediated transendocytosis of costimulatory molecules primarily targets migratory dendritic cells. Sci Immunol. 2019 May 31;4(35):eaaw0902. doi: 10.1126/sciimmunol.aaw0902. PMID: 31152091; PMCID: PMC6570622.
Kim GR, Choi JM. Current Understanding of Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) Signaling in T-Cell Biology and Disease Therapy. Mol Cells. 2022 Aug 31;45(8):513-521. doi: 10.14348/molcells.2022.2056. Epub 2022 Jul 27. PMID: 35950451; PMCID: PMC9385567.