KC-2954

293T-Siglec6-Cell-Line

26837

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Background of 293T-Siglec6-Cell-Line

Siglec6, also known as CD327, 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. Immunomodulatory Siglecs are controlled by their glycoprotein and glycolipid ligands, Siglec-6 can recognize glycolipids independent of its canonical binding pocket, suggesting that Siglec-6 possesses a secondary binding pocket tailored for recognizing glycolipids in a bilayer. The selective, stable expression and potent inhibitory capacity of Siglec-6 on human mast cells are favorable for its use as a therapeutic target in mast cell-driven diseases.

Specifications

Catalog Number	KC-2954
Cell Line Name	293T-Siglec6-Cell-Line
Host Cell Line	293T
Description	Stable HEK293T cell line 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 + 1μg/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 24-30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T SIGLEC6 cell line was generated using a lentiviral vector expressing the SIGLEC6 sequence.

Characterization

Figure: Characterization of SIGLEC6 overexpression in the 293T human SIGLEC6 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/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% 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.
Robida PA, Rische CH, Morgenstern NB, Janarthanam R, Cao Y, Krier-Burris RA, Korver W, Xu A, Luu T, Schanin J,
Leung J, Rothenberg ME, Wechsler JB, Youngblood BA, Bochner BS, O'Sullivan JA. Functional and Phenotypic
Characterization of Siglec-6 on Human Mast Cells. Cells. 2022 Mar 28;11(7):1138.
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-