KC-4958

293T-mouse-CD8a Cell Line

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Background of 293T-mouse-CD8a 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.

Specifications

Catalog Number	KC-4958
Cell Line Name	293T-mouse-CD8a Cell Line
NCBI/UniProt Accession Number	NM_001081110.2
Clone Number	4-7#
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous mouse CD8a 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

Cell Line Generation

293T-mouse-CD8a cell line was generated using lentiviral vector expressing mouse CD8a sequence.

Characterization

Figure 1: Characterization of mouse CD8a overexpression in the 293T-mouse-CD8a stable clone using PCR sequence

Figure 2: Characterization of mouse CD8a overexpression in the 293T-mouse-CD8a stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS + 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 1:4-1:5 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. 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.
2. 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.
3. Srinivasan S, Zhu C, McShan AC. Structure, function, and immunomodulation of the CD8 co-receptor. Front Immunol. 2024 Aug 26;15:1412513.

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.