KC-1719

CHOK1-Dog-CD73-Cell-Line

22679

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Background of CHOK1-Dog-CD73-Cell-Line

CD73, also known as NT5E, CD73 (ecto-5'-nucleotidase) is a novel immunoinhibitory protein that plays a key role in tumor growth and metastasis. Its main function is to convert extracellular ATP to immunosuppressive adenosine in concert with CD39 in normal tissues to limit genes. excessive immune response. Inducible expression of CD73 and other adenosinergic molecules on cancer and host cells sustains the immunosuppressive tumor microenvironment by affecting multiple aspects of the immune response. It is an ideal therapeutic target for cancer treatment, especially with conventional therapy and/or other immune checkpoint inhibitors. Research has shown that regulatory T cells can express CD73 and inhibit T cell responses via the production of adenosine.

Specifications

Catalog Number	KC-1719
Cell Line Name	CHOK1-Dog-CD73-Cell-Line
Host Cell Line	Chinese hamster ovary CHO-K1 cell line
Description	Stable CHOK1 cell line expressing exogenous Dog CD73 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% RPMI1640+20% FBS+10% DMSO
Propagation Medium	RPMI1640+10%FBS+10µg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial-like
Subculture	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-2 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 24 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

CHOK1 Dog CD73 cell line was generated using a lentiviral vector expressing the Dog CD73 sequence.

Characterization

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS + 10µg/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 1-2 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 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

Roh M, Wainwright DA, Wu JD, Wan Y, Zhang B. Targeting CD73 to augment cancer immunotherapy. Curr Opin Pharmacol. 2020 Aug;53:66-76.
Ghalamfarsa G, Kazemi MH, Raoofi Mohseni S, Masjedi A, Hojjat-Farsangi M, Azizi G, Yousefi M, Jadidi-Niaragh F. CD73 as a potential opportunity for cancer immunotherapy. Expert Opin Ther Targets. 2019 Feb;23(2):127- 142.
Neo SY, Yang Y, Record J, Ma R, Chen X, Chen Z, Tobin NP, Blake E, Seitz C, Thomas R, Wagner AK, Andersson J, de Boniface J, Bergh J, Murray S, Alici E, Childs R, Johansson M, Westerberg LS, Haglund F, Hartman J, Lundqvist A. CD73 immune checkpoint defines regulatory NK cells within the tumor microenvironment. J Clin Invest. 2020 Mar 2;130(3):1185-1198.