KC-1423

CHOK1-LILRB2-low Cell Line

22111

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Background of CHOK1-LILRB2-low Cell Line

LILRB2, also known as ILT4, is a member of the leukocyte immunoglobulin-like receptor family, which is expressed on immune cells and negatively regulates immune response by engaging class I MHC molecules on antigen-presenting cells. LILRB2 is a powerful cancer-promoting molecule that is overexpressed in leukemia, breast cancer, NSCLC and pancreatic cancer cells, and its cancer-promoting function depends on the binding of its ligand LILRB2 can induce a tolerance phenotype of human DCs, which induces the production of inhibitory T cells and inhibits the activation and proliferation of T cells.

Specifications

Catalog Number	KC-1423
Cell Line Name	CHOK1-LILRB2-low Cell Line
NCBI/UniProt Accession Number	BC036827/AAH36827.1
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous human LILRB2-Low 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 + 10ug/ml Puromycin
Selection Marker	Puromycin
Morphology	Fibroblastoid cells growing as a monolayer
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

CHOK1 LILRB2-Low cell line was generated using a lentiviral vector expressing the human LILRB2-Low sequence.

Characterization

Figure: Characterization of LILRB2-Low overexpression in CHOK1 stable clones using FACS.

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 (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.The Interaction of LILRB2 with HLA-B Is Associated with Psoriasis Susceptibility.Yanovsky RL, et al. J Invest Dermatol, 2020 Jun. PMID 31874134.
2.Immune inhibitory receptor LILRB2 is critical for the endometrial cancer progression.Shao H, et al. Biochem Biophys Res Commun, 2018 Nov 17. PMID 30343889.
3.Immunoglobulinlike transcript 4 and human leukocyte antigenG interaction promotes the progression of human colorectal cancer.Cai Z, et al. Int J Oncol, 2019 Jun. PMID 30942436.