KC-4458

293T-mouse-Epcam-Cell-Line

47103

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Background of 293T-mouse-Epcam-Cell-Line

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein functioned as cell-cell adhesion in epithelia, cell migration, proliferation and differentiation. EpCAM have oncogenic potential through upregulation of c-myc, cyclins A & E after cleavage. Epcam is not only used as diagnostic marker for various cancer, but also a potential target for cancer therapy.

Specifications

Catalog Number	KC-4458
Cell Line Name	293T-mouse-Epcam-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous mouse-Epcam 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

Cell Line Generation

293T-mouse-Epcam-cell-line was generated using a lentiviral vector expressing the mouse-Epcam sequence.

Characterization

Figure 1: Characterization of mouse-Epcam overexpression in the 293T-mouse-Epcam stable clone using qPCR.

Figure 2: Characterization of mouse-Epcam in the 293T-mouse-Epcam stable clone using PCR sequencing.

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

Litvinov, Sergey; et al. (1994). Ep-CAM: a human epithelial antigen is a homophilic cell–cell adhesion molecule. The Journal of Cell Biology. 125 (2): 437–46
Maetzel, Dorothea; et al. (2009). Nuclear signalling by tumour-associated antigen EpCAM. Nature Cell Biology. 11 (2): 162–71.
Osta, WA; et al. (2004). EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy. Cancer Res. 64 (16): 5818–24.