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	One vial 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.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.