KC-5725

CHOK1-TIM3 Cell Line

59556

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Background of CHOK1-TIM3 Cell Line

HAVCR2 (Hepatitis A Virus Cellular Receptor 2) also known as TIM3, it is a Protein Coding gene. T-cell immunoglobulin and mucin domain 3 (TIM-3) belongs to the group of inhibitory checkpoint receptors and also marks dysfunctional CD8 T cells in various kinds of cancers. T cell immunoglobulin and mucin domain 3 (TIM-3) was originally found to be expressed on the surface of Th1 cells, acting as a negative regulator and binding to the ligand galectin-9 to mediate Th1 cell the apoptosis. Studies have shown that TIM-3 plays an important role in autoimmune diseases, chronic viral infections and tumors. Diseases associated with HAVCR2 include T-Cell Lymphoma, Subcutaneous Panniculitis-Like and Hepatitis A.

Specifications

Catalog Number	KC-5725
Cell Line Name	CHOK1-TIM3 Cell Line
Clone Number	12-1#
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous human TIM3 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 1:4-1:8 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

Cell Line Generation

CHOK1 human TIM3 Cell Line was generated using a lentiviral vector expressing the human TIM3 sequence.

Characterization

Figure 1: Characterization of human TIM3 overexpression in the CHO-K1 human TIM3 stable clone using PCR sequence

Figure 2: Characterization of human TIM3 overexpression in the CHO-K1 human TIM3 stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 + 10% FBS + 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 1-2 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% 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. Wu J, Lin G, Zhu Y, Zhang H, Shi G, Shen Y, Zhu Y, Dai B, Ye D. Low TIM3 expression indicates poor prognosis of metastatic prostate cancer and acts as an independent predictor of castration resistant status. Sci Rep. 2017 Aug 21;7(1):8869.
2.Tyrinova TV, Chernykh ER. Inhibitory Checkpoint Receptor TIM-3 as a Regulator of the Functional Activity of Dendritic Cells. Bull Exp Biol Med. 2024 Jul;177(3):287-292.