KC-5467

Jurkat-CD8a-CD8b Cell Line

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Background of Jurkat-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-5467
Cell Line Name	Jurkat-CD8a-CD8b Cell Line
Clone Number	2#
Host Cell Line	Jurkat
Description	Stable Jurkat cell line 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% RPMI1640 + 20% FBS + 10% DMSO
Propagation Medium	RPMI1640 + 10% FBS + 1μg/ml Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:6 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 26 hours
Mycoplasma Status	Negative

Cell Line Generation

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

Characterization

Figure 1: Characterization of human CD8a and CD8b overexpression in the Jurkat human CD8a CD8b stable clone using PCR sequence

Figure 2: Characterization of human CD8a and CD8b overexpression in the Jurkat human CD8a CD8b stable clone using FACS.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 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 1:4-1:6 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% RPMI1640 + 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.
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.