KC-5489

293T-clv-CDCP1-Flag Cell Line

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

CDCP1, also known as CD318. This gene encodes a transmembrane protein which contains three extracellular CUB domains and acts as a substrate for 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. 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-5489
Cell Line Name	293T-clv-CDCP1-Flag Cell Line
Clone Number	7#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous clv 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 1:4-1:6 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 clv CDCP1 Flag cell line was generated using a lentiviral vector expressing the clv 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 clv CDCP1 Flag stable clone using FACS.

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

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 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 1:4-1:6 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

Huang H, Pan Y, Mai Q, Zhang C, Du Q, Liao Y, Qin S, Chen Y, Huang J, Li J, Liu T, Zou Q, Zhou Y, Yuan L, Wang W, Liang Y, Pan CY, Liu J, Yao S. Targeting CDCP1 boost CD8+ T cells-mediated cytotoxicity in cervical cancer via the JAK/STAT signaling pathway. J Immunother Cancer. 2024 Oct 24;12(10):e009416.