KC-6302

293T-siglec6-low Cell Line

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Background of 293T-siglec6-low Cell Line

SIGLEC6 (Sialic Acid Binding Ig Like Lectin 6) is a Protein Coding gene. This gene encodes a member of the SIGLEC (sialic acid binding immunoglobulin-like lectin) family of proteins. The encoded transmembrane receptor binds sialyl-TN glycans and leptin. Placental expression of the encoded protein is upregulated in preeclampsia.

Specifications

Catalog Number	KC-6302
Cell Line Name	293T-siglec6-low Cell Line
NCBI/UniProt Accession Number	NM_001245.7
Clone Number	9#
Host Cell Line	293T
Description	293T cell line stably expressing exogenous 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1 μg/mL puromycin
Selection Marker	Puromycin
Morphology	Epithelial
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

Cell Line Generation

293T-siglec6-low cell line was generated using a lentiviral vector expressing the siglec6 sequence.

Characterization

Figure1: Characterization of siglec6 overexpressing in 293T stable clones using FACS.Expression levels were quantified by qFACS at approximately 11198 molecules per cell.

Figure2: Characterization of 293T-siglec6-low cell line stable clone using PCR sequencing.

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS + 1 μg/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 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 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

Schmidt EN, Lamprinaki D, McCord KA, Joe M, Sojitra M, Waldow A, Nguyen J, Monyror J, Kitova EN, Mozaneh F, Guo XY, Jung J, Enterina JR, Daskhan GC, Han L, Krysler AR, Cromwell CR, Hubbard BP, West LJ, Kulka M, Sipione S, Klassen JS, Derda R, Lowary TL, Mahal LK, Riddell MR, Macauley MS. Siglec-6 mediates the uptake of extracellular vesicles through a noncanonical glycolipid binding pocket. Nat Commun. 2023 Apr 22;14(1):2327. doi: 10.1038/s41467-023-38030-6. PMID: 37087495; PMCID: PMC10122656.
Guan X, Yu M, Wu L, Chen J, Tong J, Wu X, Yin A, Xiao T, Wang B, Zhang JV, Niu J. Elevated trophoblastic Siglec6 contributes to the impairment of vascular endothelial cell functions by downregulating Wnt6/β-catenin signaling in preeclampsia. Arch Biochem Biophys. 2022 Nov 15;730:109396. doi: 10.1016/j.abb.2022.109396. Epub 2022 Sep 14. PMID: 36113626.
Smiljkovic D, Herrmann H, Sadovnik I, Gamperl S, Berger D, Stefanzl G, Eisenwort G, Hoermann G, Kopanja S, Dorofeeva Y, Focke-Tejkl M, Jaksch P, Hoetzenecker K, Szepfalusi Z, Valenta R, Arock M, Valent P. Expression and regulation of Siglec-6 (CD327) on human mast cells and basophils. J Allergy Clin Immunol. 2023 Jan;151(1):202-211. doi: 10.1016/j.jaci.2022.07.018. Epub 2022 Aug 8. PMID: 35953001.