KC-4623

293T-CD8a-CD8b Cell Line

55280

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Background of 293T-CD8a-CD8b Cell Line

The CD8 glycoprotein is expressed by thymocytes, mature T cells and natural killer (NK) cells and has been implicated in the recognition of monomorphic determinants on major histocompatibility complex (MHC) Class I antigens, and in signal transduction during the course of T-cell activation. The CD8 glycoprotein is comprised of two distinct polypeptide chains, alpha and beta, which have the capacity to form homodimeric (CD8 alpha/alpha) or heterodimeric (CD8 alpha/beta) cell surface complexes. 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.
As an important member of T cytotoxic pathway-related genes, CD8a molecule (CD8A) may be a useful biomarker of immunotherapeutic response and immune cell infiltration. The demonstrates that a CD8+ T-cell clone can express both CD8 alpha/alpha homodimers and CD8 alpha/beta heterodimers and these two complexes do not have identical biological properties. CD8 alpha/alpha and CD8 alpha/beta dimers may not subserve identical functions.

Specifications

Catalog Number	KC-4623
Cell Line Name	293T-CD8a-CD8b Cell Line
Clone Number	4#
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous human CD8a and 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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

293T-CD8a-CD8b cell line was generated using a lentiviral vector expressing the human CD8a and CD8b sequence.

Characterization

Figure 1: Characterization of CD8a and CD8b overexpression in the 293T stable clone using FACS(Kyinno, Cat#KB-1284).

Figure 2: Characterization of CD8a and CD8b in the 293T stable clone using PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/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 9.0mL 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 1:4-1:8 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. Zheng Z, Guo Y, Huang X, Liu J, Wang R, Qiu X, Liu S. CD8A as a Prognostic and Immunotherapy Predictive Biomarker Can Be Evaluated by MRI Radiomics Features in Bladder Cancer. Cancers (Basel). 2022 Oct 5;14(19):4866. doi: 10.3390/cancers14194866. PMID: 36230788; PMCID: PMC9564077.
2. Giblin PA, Leahy DJ, Mennone J, Kavathas PB. The role of charge and multiple faces of the CD8 alpha/alpha homodimer in binding to major histocompatibility complex class I molecules: support for a bivalent model. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1716-20. doi: 10.1073/pnas.91.5.1716. PMID: 8127870; PMCID: PMC43234.
3. Terry LA, DiSanto JP, Small TN, Flomenberg N. Differential expression and regulation of the human CD8 alpha and CD8 beta chains. Tissue Antigens. 1990 Feb;35(2):82-91. doi: 10.1111/j.1399-0039.1990.tb01761.x. PMID: 2111591.
4. Srinivasan S, Zhu C, McShan AC. Structure, function, and immunomodulation of the CD8 co-receptor. Front Immunol. 2024 Aug 26;15:1412513. doi: 10.3389/fimmu.2024.1412513. PMID: 39253084; PMCID: PMC11381289.