KC-4509

NCI-H1299-Luc2-CPNE1-Flag-Cell-Line

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Background of NCI-H1299-Luc2-CPNE1-Flag-Cell-Line

Calcium-dependent membrane-binding proteins may regulate molecular events at the interface of the cell membrane and cytoplasm. This gene encodes a calcium-dependent protein that also contains two N-terminal type II C2 domains and an integrin A domain-like sequence in the C-terminus. However, the encoded protein does not contain a predicted signal sequence or transmembrane domains. This protein has a broad tissue distribution and it may function in membrane trafficking. This gene and the gene for RNA binding motif protein 12 overlap at map location 20q11.21. Alternate splicing results in multiple transcript variants encoding different proteins.

Specifications

Catalog Number	KC-4509
Cell Line Name	NCI-H1299-Luc2-CPNE1-Flag-Cell-Line
Host Cell Line	NCI-H1299-Luc2
Description	Stable NCI H1299 Luc2 clone expressing exogenous CPNE1 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 + 1μg/ml Puromycin+300μg/ml Hygromycin B
Selection Marker	Puromycin, Hygromycin B
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:4 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

NCI H1299 Luc2 CPNE1 Flag Cell Line was generated using a lentiviral vector expressing the CPNE1 sequence.

Characterization

Figure 1: Characterization of CPNE1 overexpression in the NCI H1299 Luc2 CPNE1 Flag stable clone using FACS.

Figure 2: Characterization of CPNE1 expression in the NCI H1299 Luc2 CPNE1 Flag stable clone using PCR sequence.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 1μg/mL Puromycin and 300μg/mL Hygromycin B)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 1:3-1:4 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 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

Zhu S, Li R, Yin K, Wu L. CPNE1, A Potential Therapeutic Target in Nasopharyngeal Carcinoma, Affects Cell Growth and Radiation Resistance. Radiat Res. 2024 Apr 1;201(4):310-316.
Shao Z, Ma X, Zhang Y, Sun Y, Lv W, He K, Xia R, Wang P, Gao X. CPNE1 predicts poor prognosis and promotes tumorigenesis and radioresistance via the AKT singling pathway in triple-negative breast cancer. Mol Carcinog. 2020 May;59(5):533-544.