KC-4499

CHOK1-cyno-siglec6 Cell Line

47395

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Background of CHOK1-cyno-siglec6 Cell Line

siglec6, also known as CD327. Sialic-acid-binding immunoglobulin-type lectins (Siglecs) are a family of cell-surface immunomodulatory receptors that recognize sialic-acid-containing glycans. The majority of Siglecs have an inhibitory motif in their intercellular domain and can regulate the cellular activation of immune cells. Importantly, the immunomodulatory role of Siglecs is regulated by engagement with distinct sialoglycan ligands. Research has described that a role for Siglec-6 in migration and adhesion of CLL B cells to CLL- bone marrow stromal cells (BMSCs) in vitro and compromised migration to bone marrow and spleen in vivo.

Specifications

Catalog Number	KC-4499
Cell Line Name	CHOK1-cyno-siglec6 Cell Line
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous cyno siglec6 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

Cell Line Generation

CHOK1 cyno siglec6 Cell Line was generated using a lentiviral vector expressing the cyno siglec6 sequence.

Characterization

Figure 1: Characterization of cyno siglec6 overexpression in the CHOK1 cyno siglec6 stable clone using qPCR.

Figure 2: Characterization of cyno siglec6 overexpression in the CHOK1 stable clone using DNA sequencing.

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 1:4-1:5 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

Jame-Chenarboo Z, Gray TE, Macauley MS. Advances in understanding and exploiting Siglec-glycan interactions. Curr Opin Chem Biol. 2024 Jun;80:102454. doi: 10.1016/j.cbpa.2024.102454. Epub 2024 Apr 16. PMID: 38631213.
Nunes J, Tafesse R, Mao C, Purcell M, Mo X, Zhang L, Long M, Cyr MG, Rader C, Muthusamy N. Siglec-6 as a therapeutic target for cell migration and adhesion in chronic lymphocytic leukemia. Nat Commun. 2024 Jun 18;15(1):5180. doi: 10.1038/s41467-024-48678-3. PMID: 38890323; PMCID: PMC11189495.