KC-3358

CT26-ENPP3 Cell Line

34348

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Background of CT26-ENPP3 Cell Line

Ecto-nucleotide pyrophosphatase-phosphodiesterase 3 (E-NPP3), also known as CD203c, it is an ATP-hydrolyzing glycoprotein that is located in the extracellular space. Expression of this protein has been detected in the uterus, basophils, and mast cells. ENPP3 levels undergo cyclic changes in the endometrium and affect embryo adhesion and invasion via altering the expression of implantation factors in the human endometrium. Therefore, ENPP3 may play an important role in embryo implantation.

Specifications

Catalog Number	KC-3358
Cell Line Name	CT26-ENPP3 Cell Line
Clone Number	5#
Host Cell Line	CT26
Description	Stable CT26 clone expressing exogenous 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 10μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Fibroblastoid
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
In Vivo Validation	NA

Cell Line Generation

CT26 ENPP3 cell line was generated using a lentiviral vector expressing the ENPP3 sequence.

Characterization

Figure 1: Characterization of ENPP3 overexpression in the CT26 ENPP3 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/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 1:4-1:8 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

Döhler C, Zebisch M, Krinke D, Robitzki A, Sträter N. Crystallization of ectonucleotide phosphodiesterase/ pyrophosphatase-3 and orientation of the SMB domains in the full-length ectodomain. Acta Crystallogr F Struct Biol Commun. 2018 Nov 1.
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.
Kabashima K, Nakashima C, Nonomura Y, Otsuka A, Cardamone C, Parente R, De Feo G, Triggiani M. Biomarkers for evaluation of mast cell and basophil activation. Immunol Rev. 2018 Mar.