KC-3715

CHOK1-EPHB4 Cell Line

34650

Home » 细胞系 » CHOK1-EPHB4 Cell Line

Background of CHOK1-EPHB4 Cell Line

EphB4 is a member of the Eph receptor family, short for Ephrin-B receptor 4, a group of receptor tyrosine kinases involved in a variety of cellular processes including intercellular communication, tissue development and cytoskeletal regulation. EphB4 plays a key role in angiogenesis, cell migration, and tissue remodeling, making it an important target for scientific research and potential drug development. EphB4 inhibitors work by interfering with signaling pathways associated with the EphB4 receptor.

Specifications

Catalog Number	KC-3715
Cell Line Name	CHOK1-EPHB4 Cell Line
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous EPHB4 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	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

CHOK1 EPHB4 Cell Line was generated using a lentiviral vector expressing the EPHB4 sequence.

Characterization

Figure 1: Characterization of EPHB4 overexpression in the CHOK1 EPHB4 stable clone using FACS.

Figure 2: Characterization of human EPHB4 overexpression in the CHO-K1 human EPHB4 stable clone using PCR sequence.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/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 (RPMI1640 + 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. Piffko A, Uhl C, Vajkoczy P, Czabanka M, Broggini T. EphrinB2-EphB4 Signaling in Neurooncological Disease. Int J Mol Sci. 2022 Jan 31;23(3):1679. doi: 10.3390/ijms23031679. PMID: 35163601; PMCID: PMC8836162. 2. Liu X, Wang K, Hou S, Jiang Q, Ma C, Zhao Q, Kong L, Chen J, Wang Z, Zhang H, Yuan T, Li Y, Huan Y, Shen Z, Hu Z, Huang Z, Cui B, Li P. Insulin induces insulin receptor degradation in the liver through EphB4. Nat Metab. 2022 Sep;4(9):1202-1213. doi: 10.1038/s42255-022-00634-5. Epub 2022 Sep 21. PMID: 36131205.. 3. Bhatia S, Nguyen D, Darragh LB, Van Court B, Sharma J, Knitz MW, Piper M, Bukkapatnam S, Gadwa J, Bickett TE, Bhuvane S, Corbo S, Wu B, Lee Y, Fujita M, Joshi M, Heasley LE, Ferris RL, Rodriguez O, Albanese C, Kapoor M, Pasquale EB, Karam SD. EphB4 and ephrinB2 act in opposition in the head and neck tumor microenvironment. Nat Commun. 2022 Jun 20;13(1):3535. doi: 10.1038/s41467-022-31124-7. PMID: 35725568; PMCID: PMC9209511.