KC-1105

CHOK1-SIGLEC7 Cell Line

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Background of CHOK1-SIGLEC7 Cell Line

SIGLEC7, also known as CD328. Siglec-7 is constitutively expressed on human eosinophils and downmodulates eosinophil activation. Targeting of Siglec-7 on eosinophils might enhance treatment efficacy in eosinophil-driven disorders. Research has shown that Siglec-7 could be utilized as a potential marker of NK cell dysfunction in acute myeloid leukemia(AML).

Specifications

Catalog Number	KC-1105
Cell Line Name	CHOK1-SIGLEC7 Cell Line
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous SIGLEC7 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+5μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:6 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 SIGLEC7 Cell Line was generated using a lentiviral vector expressing the SIGLEC7 sequence.

Characterization

Figure 1: Characterization of endogenous SIGLEC7 expression in CHOK1 cell using FACS.

Figure 2: Characterization of SIGLEC7 overexpression in the CHOK1 SIGLEC7 stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (F12K supplemented with 10% FBS and 5μg/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 1:4-1:6 every 2-3 days; seed out at about 1-3 × 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. Legrand F, Landolina N, Zaffran I, Emeh RO, Chen E, Klion AD, Levi-Schaffer F. Siglec-7 on peripheral blood eosinophils: Surface expression and function. Allergy. 2019 Jul;74(7):1257-1265. doi: 10.1111/all.13730. Epub 2019 Feb 26. PMID: 30690753; PMCID: PMC6661208.
2. Yang L, Feng Y, Wang S, Jiang S, Tao L, Li J, Wang X. Siglec-7 is an indicator of natural killer cell function in acute myeloid leukemia. Int Immunopharmacol. 2021 Oct;99:107965. doi: 10.1016/j.intimp.2021.107965. Epub 2021 Jul 14. PMID: 34273636.