KC-5059

293T-N-CDCP1-Flag Cell Line

59051

Home » 293T-N-CDCP1-Flag Cell Line

Background of 293T-N-CDCP1-Flag Cell Line

CUB-domain containing protein 1 (CDCP1) is a type I transmembrane glycoprotein, it is not only a surface marker for cells with higher metastatic potential, but also functionally involved in enhancing tumor metastasis. It is upregulated in malignancies of the breast, lung, colorectum, ovary, kidney, liver, pancreas, and hematopoietic system. CUB domain-containing protein 1 (CDCP1) is a substrate of Src family kinases. The protein plays a role in the tyrosine phosphorylation-dependent regulation of cellular events that are involved in tumor invasion and metastasis. Knockdown of CDCP1 blocks tumor metastasis or peritoneal dissemination in vivo, without significantly affecting cell proliferation. Research have shown that CDCP1 plays a critical role in cervical cancer progression by modulating the tumor immune microenvironment. Targeting CDCP1 offers a promising therapeutic strategy to improve the outcome of patients with cervical cancer.

Specifications

Catalog Number	KC-5059
Cell Line Name	293T-N-CDCP1-Flag Cell Line
Clone Number	1-8#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous N CDCP1 Flag 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-N-CDCP1-Flag cell line was generated using a lentiviral vector expressing the N CDCP1 Flag sequence.

Characterization

Figure 1: Characterization of endogenous Flag tag expression in 293T cell using FACS.

Figure 2: Characterization of Flag tag overexpression in the 293T-N-CDCP1-Flag stable clone using FACS.

Figure 3: Characterization of N CDCP1 in the 293T-N-CDCP1-Flag stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 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. Liu H, Ong SE, Badu-Nkansah K, Schindler J, White FM, Hynes RO. CUB-domain-containing protein 1 (CDCP1) activates Src to promote melanoma metastasis. Proc Natl Acad Sci U S A. 2011 Jan 25;108(4):1379-84. doi: 10.1073/pnas.1017228108. Epub 2011 Jan 10. PMID: 21220330; PMCID: PMC3029734.
2. Qi X, Gao J, Li Z, Zhang G, Li J, Fu Y, Cai M, Wang H, Tong T. CDCP1: A promising diagnostic biomarker and therapeutic target for human cancer. Life Sci. 2022 Jul 15;301:120600. doi: 10.1016/j.lfs.2022.120600. Epub 2022 May 2. PMID: 35504333.
3. Khan T, Kryza T, Lyons NJ, He Y, Hooper JD. The CDCP1 Signaling Hub: A Target for Cancer Detection and Therapeutic Intervention. Cancer Res. 2021 May 1;81(9):2259-2269. doi: 10.1158/0008-5472.CAN-20-2978. Epub 2021 Jan 28. PMID: 33509939.
4. Uekita T, Sakai R. Roles of CUB domain-containing protein 1 signaling in cancer invasion and metastasis. Cancer Sci. 2011 Nov;102(11):1943-8. doi: 10.1111/j.1349-7006.2011.02052.x. Epub 2011 Sep 6. PMID: 21812858.