KC-3251

CT26-luc2-CEA6-CEA5-Cell-Line

34254

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Background of CT26-luc2-CEA6-CEA5-Cell-Line

CEACAM5 and CEACAM6, are also known as CD66e and CD66c. CEACAM5 is a cell surface glycoprotein that represents the founding member of the carcinoembryonic antigen (CEA) family of proteins. It takes part in cell adhesion and intracellular signaling as a cell surface glycoprotein, and also plays a role as an oncogene by promoting tumor progression, inducing resistance to anoikis of colorectal carcinoma cells. Furthermore, CEACAM5 mediates homophilic and heterophilic cell adhesion with other carcinoembryonic antigen-related cell adhesion molecules, such as CEACAM6. CEACAM6 is a cell surface glycoprotein that belongs to the CEA family whose members are glycosyl phosphatidyl inositol anchored cell surface glycoproteins. CEACAM6 plays a role in cell adhesion and is widely used as tumor markers in serum immunoassay determinations of carcinoma. CEACAM6 affects the sensitivity of tumor cells to adenovirus infection, and acts as a receptor for adherent-invasive E. coli adhesion to the surface of ileal epithelial cells in patients with Crohn's disease.

Specifications

Catalog Number	KC-3251
Cell Line Name	CT26-luc2-CEA6-CEA5-Cell-Line
Host Cell Line	CT26
Description	Stable CT26 cell line expressing exogenous luciferase under the control of CEACAM6-CEACAM5 signaling pathway.
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 + 600μg/mL Hygromycin B + 10μg/mL Puromycin
Selection Marker	Hygromycin B, Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1 : 4~1 : 8 every 3 days; seed out at about 1 × 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

CT26-Luc2-CEACAM6-CEACAM5 cell line was generated using a lentiviral vector expressing the luciferase, CEACAM5 and CEACAM6 sequence.

Characterization

Figure1: Characterization of CEACAM5 overexpression in CT26 stable clones using FACS.

Figure2: Characterization of CEACAM6 overexpression in CT26 stable clones using FACS.

Figure3: Characterization of CT26-Luc2-CEACAM6-CEACAM5 cell line stable clone using luciferase assay in the conditions of different cell numbers.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS, 600µg/mL Hygromycin B + 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 3 days; seed out at about 1 × 10⁵ cells/mL

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

1. Entrez Gene: CEACAM6 carcinoembryonic antigen-related cell adhesion molecule 6 (non-specific cross reacting antigen).
2. Human PubMed Reference: National Center for Biotechnology Information, U.S. National Library of Medicine.
3. Benchimol S, Fuks A, Jothy S, Beauchemin N, Shirota K, Stanners CP. Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule. Cell. 1989, 4, 57(2): 327–334.
4. Ilantzis C, DeMarte L, Screaton RA, Stanners CP. Deregulated expression of the human tumor marker CEA and CEA family member CEACAM6 disrupts tissue architecture and blocks colonocyte differentiation. Neoplasia. 4(2): 151–163.
5. Ordoñez C, Screaton RA, Ilantzis C, Stanners CP. Human carcinoembryonic antigen functions as a general inhibitor of anoikis. Cancer Research. 2000, July, 60 (13): 3419–3424.