KC-5117

293T-cyno-CD58 Cell Line

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Background of 293T-cyno-CD58 Cell Line

This gene encodes a member of the immunoglobulin superfamily. The encoded protein is a ligand of the T lymphocyte CD2 protein, and functions in adhesion and activation of T lymphocytes. The protein is localized to the plasma membrane. Alternatively spliced transcript variants have been described.

Specifications

Catalog Number	KC-5117
Cell Line Name	293T-cyno-CD58 Cell Line
Clone Number	1#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous cyno-CD58 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

Cell Line Generation

293T-cyno-CD58 cell line was generated using a lentiviral vector expressing the cyno-CD58 sequence.

Characterization

Figure 1: Characterization of cyno-CD58 overexpression in the 293T-cyno-CD58 stable clone using FACS.

Figure 2: Characterization of cyno-CD58 overexpressing in 293T-cyno-CD58 stable clone using PCR sequencing.

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

Abdul Razak FR, Diepstra A, Visser L, van den Berg A. CD58 mutations are common in Hodgkin lymphoma cell lines and loss of CD58 expression in tumor cells occurs in Hodgkin lymphoma patients who relapse. Genes Immun. 2016 Sep;17(6):363-6. doi: 10.1038/gene.2016.30. Epub 2016 Jul 28. PMID: 27467287.
Yamamoto M, Watanabe M, Inoue N, Watanabe A, Ozaki H, Ohsaki M, Hidaka Y, Iwatani Y. Association of CD58 Polymorphisms and its Protein Expression with the Development and Prognosis of Autoimmune Thyroid Diseases. Immunol Invest. 2020 Feb;49(1-2):106-119. doi: 10.1080/08820139.2019.1659811. Epub 2019 Sep 11. PMID: 31505972.