KC-5705

CHOK1-mouse-CD8a-CD8b Cell Line

59661

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Background of CHOK1-mouse-CD8a-CD8b Cell Line

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.
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, acting as a coreceptor, and the T-cell receptor on the T lymphocyte recognize antigens displayed by an antigen presenting cell (APC) in the context of class I MHC molecules. The functional coreceptor is either a homodimer composed of two alpha chains, or 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 beta chain isoforms. Multiple alternatively spliced transcript variants encoding distinct membrane associated or secreted isoforms have been described. A pseudogene, also located on chromosome 2, has been identified.

Specifications

Catalog Number	KC-5705
Cell Line Name	CHOK1-mouse-CD8a-CD8b Cell Line
Clone Number	7#
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous mouse-CD8a and mouse-CD8b 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640+10%FBS+10μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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

CHOK1-mouse-CD8a-CD8b Cell Line was generated using a lentiviral vector expressing the mouse-CD8a and mouse-CD8b sequence.

Characterization

Figure 1: Characterization of mouse-CD8a overexpression in the CHOK1-mouse-CD8a-CD8b stable clone using FACS.

Figure 2: Characterization of mouse-CD8b overexpression in the CHOK1-mouse-CD8a-CD8b stable clone using FACS.

Figure 3: Characterization of mouse-CD8a and mouse-CD8b overexpression in the CHOK1-mouse-CD8a-CD8b stable clone using PCR.

Cell Resuscitation

Prewarm culture medium (RPMI1640+10%FBS+10μg/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 1:4-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

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.
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.
Shiue L, Gorman SD, Parnes JR. A second chain of human CD8 is expressed on peripheral blood lymphocytes. J Exp Med. 1988 Dec 1;168(6):1993-2005. doi: 10.1084/jem.168.6.1993. PMID: 3264320; PMCID: PMC2189163.
Thakral D, Dobbins J, Devine L, Kavathas PB. Differential expression of the human CD8beta splice variants and regulation of the M-2 isoform by ubiquitination. J Immunol. 2008 Jun 1;180(11):7431-42. doi: 10.4049/jimmunol.180.11.7431. PMID: 18490743.