KC-4866

293T-cyno-CD8a Cell Line

52793

Home » 293T-cyno-CD8a Cell Line

Background of 293T-cyno-CD8a Cell Line

CD8A (CD8 Subunit Alpha) is a Protein Coding gene. The CD8 antigen is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates efficient cell-cell interactions within the immune system. The CD8 antigen acts as a coreceptor with the T-cell receptor on the T lymphocyte to recognize antigens displayed by an antigen presenting cell in the context of class I MHC molecules. The coreceptor functions as either a homodimer composed of two alpha chains or as a heterodimer composed of one alpha and one beta chain. Both alpha and beta chains share significant homology to immunoglobulin variable light chains. This gene encodes the CD8 alpha chain. Multiple transcript variants encoding different isoforms have been found for this gene. The major protein isoforms of this gene differ by the presence or absence of a transmembrane domain and thus differ in being a membrane-anchored or secreted protein.

Specifications

Catalog Number	KC-4866
Cell Line Name	293T-cyno-CD8a Cell Line
Host Cell Line	293T
Description	293T cell line stably expressing exogenous cyno CD8a 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	Fibroblastoid cells growing as monolayer
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-CD8a cell line was generated using a lentiviral vector expressing the cyno CD8a sequence.

Characterization

Figure1: Characterization of cyno CD8a overexpression in 293T cyno CD8a stable clones using quantitative real-time PCR.

Figure2: Characterization of 293T cyno CD8a cell line stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1 μg/mL puromycin) in a 37°C water bath.
2. Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
3. Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
4. Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0 mL complete culture medium.
5. Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
6. Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
7. Incubate the flask at 37°C, 5% CO₂ incubator.
8. Split saturated culture 1:4-1:5 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 20% FBS + 10% DMSO) fresh immediately before use.
2. Keep the freezing medium on ice and label cryovials.
3. Transfer cells to a sterile, conical centrifuge tube, and count the cells.
4. Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
5. Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
6. Aliquot 1 mL of the cell suspension into each cryovial.
7. Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
8. Transfer vials to liquid nitrogen for long-term storage.

References

1.Snow PM, Terhorst C. The T8 antigen is a multimeric complex of two distinct subunits on human thymocytes but consists of homomultimeric forms on peripheral blood T lymphocytes. J Biol Chem. 1983 Dec 10;258(23):14675-81. PMID: 6605969.
2.Mancebo E, Moreno-Pelayo MA, Mencía A, de la Calle-Martín O, Allende LM, Sivadorai P, Kalaydjieva L, Bertranpetit J, Coto E, Calleja-Antolín S, Ruiz-Contreras J, Paz-Artal E. Gly111Ser mutation in CD8A gene causing CD8 immunodeficiency is found in Spanish Gypsies. Mol Immunol. 2008 Jan;45(2):479-84. doi: 10.1016/j.molimm.2007.05.022. Epub 2007 Jul 20. PMID: 17658607.