KC-2265

293T-Siglec8-Cell-Line

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Background of 293T-Siglec8-Cell-Line

Sialic acid-binding immunoglobulin (Ig)-like lectins, or SIGLECs, are a family of type 1 transmembrane proteins each having a unique expression pattern, mostly in hemopoietic cells. SIGLEC8 is a member of the CD33-like subgroup of SIGLECs, which are localized to 19q13.3-q13.4 and have 2 conserved cytoplasmic tyrosine-based motifs: an immunoreceptor tyrosine-based inhibitory motif, or ITIM, and a motif homologous to one identified in signaling lymphocyte activation molecule that mediates an association with SLAM-associated protein.Diseases associated with SIGLEC8 include Eosinophilic Gastritis and Chronic Inducible Urticaria.

Specifications

Catalog Number	KC-2265
Cell Line Name	293T-Siglec8-Cell-Line
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous Siglec8 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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T-Siglec8 cell line was generated using a lentiviral vector expressing the Siglec8 sequence.

Characterization

Figure 1: Characterization of Siglec8 overexpression in the 293T Siglec8 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

Foussias G, Yousef GM, Diamandis EP. Molecular characterization of a Siglec8 variant containing cytoplasmic tyrosine-based motifs, and mapping of the Siglec8 gene. Biochem Biophys Res Commun. 2000 Nov 30;278(3):775-81. doi: 10.1006/bbrc.2000.3866. PMID: 11095983.
Trebo A, Ditsch N, Degenhardt T, Kuhn C, Rahmeh M, Schmoeckel E, Mayr D, Czogalla B, Kolben T, Meister S, Mahner S, Jeschke U, Hester A. First Evidence for a Role of Siglec-8 in Breast Cancer. Int J Mol Sci. 2021 Feb 18;22(4):2000. doi: 10.3390/ijms22042000. PMID: 33670444; PMCID: PMC7922794.
Sajay-Asbaghi M, Sadeghi-Shabestrai M, Monfaredan A, Seyfizadeh N, Razavi A, Kazemi T. Promoter region single nucleotide polymorphism of siglec-8 gene associates with susceptibility to allergic asthma. Per Med. 2020 May 1;17(3):195-201. doi: 10.2217/pme-2018-0080. Epub 2020 Feb 20. PMID: 32077788.