KC-2693

4T1-Epcam-Cell-Line

26759

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Background of 4T1-Epcam-Cell-Line

Epcam, also known as MK-1. This gene encodes a carcinoma-associated antigen and is a member of a family that includes at least two type I membrane proteins. This antigen is expressed on most normal epithelial cells and gastrointestinal carcinomas and functions as a homotypic calcium-independent cell adhesion molecule. The antigen is being used as a target for immunotherapy treatment of human carcinomas. Mutations in this gene result in congenital tufting enteropathy. A combination of CK19, EpCAM and CEACAM5 gene expressions in lymph nodes can be used to identify micrometastasis which postoperatively may be used as a tool to predict patients' recurrence and survival.

Specifications

Catalog Number	KC-2693
Cell Line Name	4T1-Epcam-Cell-Line
Host Cell Line	4T1
Description	Stable 4T1 cell line expressing exogenous 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 4μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:6-1:8 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 12.6 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

4T1 Epcam Cell Line was generated using a lentiviral vector expressing the Epcam sequence.

Characterization

Figure 1: Characterization of Epcam overexpression in the 4T1 Epcam stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 4μg/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 1:6-1:8 every 2-3 days; seed out at about 1-3 × 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

Süer H, Erus S, Cesur EE, Yavuz Ö, Ağcaoğlu O, Bulutay P, Önder TT, Tanju S, Dilege Ş. Combination of CEACAM5, EpCAM and CK19 gene expressions in mediastinal lymph node micrometastasis is a prognostic factor for non-small cell lung cancer. J Cardiothorac Surg. 2023 Jun 13;18(1):189. doi: 10.1186/s13019-023-02297-z. PMID: 37312199; PMCID: PMC10262366.