KC-2742

293T-cyno-ENPP3 Cell Line

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Background of 293T-cyno-ENPP3 Cell Line

Exonucleotide pyrophosphatase-phosphodiesterase 3 (ENPP3) is a protein detected in the womb that plays an important role in tumor development and invasion. ENPP3 is expressed in human endometrial epithelial cells, and its expression level affects the menstrual cycle and affects embryo adhesion and invasion by altering the expression of implantation factors in the human endometrial. Therefore, ENPP3 may play an important role in embryo implantation.

Specifications

Catalog Number	KC-2742
Cell Line Name	293T-cyno-ENPP3 Cell Line
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous cyno-ENPP3 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 1:4-1:5 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
In Vivo Validation	NA

Cell Line Generation

293T-cyno-ENPP3 cell line was generated using a lentiviral vector expressing the cyno-ENPP3 sequence.

Characterization

Figure 1: Characterization of cyno-ENPP3 overexpression in the 293T-cyno-ENPP3 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 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 1:4-1:5 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

Chen Q, Xin A, Qu R, Zhang W, Li L, Chen J, Lu X, Gu Y, Li J, Sun X. Expression of ENPP3 in human cyclic endometrium: a novel molecule involved in embryo implantation. Reprod Fertil Dev. 2018 Oct;30(10):1277-1285. doi: 10.1071/RD17257. PMID: 29614240.
Doñate F, Raitano A, Morrison K, An Z, Capo L, Aviña H, Karki S, Morrison K, Yang P, Ou J, Moriya R, Shostak Y, Malik F, Nadell R, Liu W, Satpayev D, Atkinson J, Joseph IB, Pereira DS, Challita-Eid PM, Stover DR. AGS16F Is a Novel Antibody Drug Conjugate Directed against ENPP3 for the Treatment of Renal Cell Carcinoma. Clin Cancer Res. 2016 Apr 15;22(8):1989-99. doi: 10.1158/1078-0432.CCR-15-1542. Epub 2015 Nov 20. PMID: 26589436.