KC-2205

HEK293-CTLA4-Δ23-Cell-Line

23594

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Background of HEK293-CTLA4-Δ23-Cell-Line

Cytotoxic T lymphocyte-associated protein 4 (CTLA4), also named CD152, is a membrane protein receptor acting as a negative regulator of T cell responsive when bound to its ligands on the surface of antigen-presenting cells, including CD80 and CD86. The dysfunction and overactivation of CTLA4 has been proved to lead to immune dysfunction syndrome and tumor immunological escape, its monoclonal antibody, Ipilimumab, is the first approved immune checkpoint blockade therapy and has been proved to be a potential therapeutically strategy for cancer patient.

Specifications

Catalog Number	KC-2205
Cell Line Name	HEK293-CTLA4-Δ23-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	Stable HEK293 cell line expressing exogenous human CTLA4-Δ23 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 1:3-1:6 every 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

HET293 human CTLA4-Δ23 cell line was generated using a lentiviral vector expressing the human CTLA4-Δ23 sequence.

Characterization

Cell Resuscitation

Prewarm culture medium (DMEM + 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 1:3-1:6 every 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 Allen, Eliezer M, Diana Miao, Bastian Schilling, Sachet A Shukla, Christian Blank, Lisa Zimmer, Antje Sucker, et al. 2015. ¡ùGenomic Correlates of Response to CTLA-4 Blockade in Metastatic Melanoma.¡ì Science 350 (6257). American Association for the Advancement of Science: 207Ã¿11.
Finn, P W, H He, Y Wang, Z Wang, G Guan, J Listman, and D L Perkins. 1997. ¡ùSynergistic Induction of CTLA-4 Expression by Costimulation with TCR Plus CD28 Signals Mediated by Increased Transcription and Messenger Ribonucleic Acid Stability.¡ì The Journal of Immunology 158 (9): 4074Ã¿81.