KC-1031

CHOK1-INSR

21413

Home » CHOK1-INSR

Background of CHOK1-INSR

INSR is a transmembrane receptor belonging to tyrosine kinase family, INSR is acttvated by insulin, IGF1, IGFII, which play an important role in the regulatton of glucose hemostasis, a functtonal process that degenerate condittons may result in a range of clinical manifestattons including diabetes and cancer.

Specifications

Catalog Number	KC-1031
Cell Line Name	CHOK1-INSR
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line stable expressing exogenous human INSR 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 + 6μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-2 × 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 human INSR cell line was generated using lenttviral vector expressing human INSR sequence.

Characterization

Figure: Characterization of INSR overexpressing in the CHOK1 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (F12K supplemented with 10% FBS and 6μg/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 1-2 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% F12K + 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. Zhande R, Mitchell JJ, Wu J, Sun XJ. Molecular mechanism of insulin-induced degradation of insulin receptor substrate 1. Mol Cell Biol. 2002 Feb;22(4):1016-26. doi: 10.1128/MCB.22.4.1016-1026.2002. PMID: 11809794; PMCID: PMC134643.
2. Sun XJ, Goldberg JL, Qiao LY, Mitchell JJ. Insulin-induced insulin receptor substrate-1 degradation is mediated by the proteasome degradation pathway. Diabetes. 1999 Jul;48(7):1359-64. doi: 10.2337/diabetes.48.7.1359. PMID: 10389839.
3. Sommerfeld MR, Müller G, Tschank G, Seipke G, Habermann P, Kurrle R, Tennagels N. In vitro metabolic and mitogenic signaling of insulin glargine and its metabolites. PLoS One. 2010 Mar 4;5(3):e9540. doi: 10.1371/journal.pone.0009540. PMID: 20209060; PMCID: PMC2832019.