KC-2701

S180-Epcam-Midddle Cell Line

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Background of S180-Epcam-Midddle 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-2701
Cell Line Name	S180-Epcam-Midddle Cell Line
Host Cell Line	Mouse S180 cell line
Description	Stable S180 cell line expressing exogenous human Epcam gene in middle level
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 + 2μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Rounded
Subculture	Split saturated culture 1:2-1:3 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 14-20 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

S180-Epcam-Middle cell Line was generated using a retroviral vector expressing human Epcam sequence.

Characterization

Figure 1: Characterization of human Epcam overexpression in the S180-Epcam stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 2μg/mL Puromycin)in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:2-1:3 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1. Litvinov, Sergey; et al. (1994). Ep-CAM: a human epithelial antigen is a homophilic cellÿcell adhesion molecule. The Journal of Cell Biology. 125 (2): 437ÿ46. 2. Maetzel, Dorothea; et al. (2009). Nuclear signalling by tumour-associated antigen EpCAM. Nature Cell Biology. 11 (2): 162ÿ71. 3. 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): 5818ÿ24.
2. Gaber A, Lenarčič B, Pavšič M. Current View on EpCAM Structural Biology. Cells. 2020 May 31;9(6):1361. doi: 10.3390/cells9061361. PMID: 32486423; PMCID: PMC7349879.