KC-5499

CHOK1-CD8a-CD8b Cell Line

56994

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Background of CHOK1-CD8a-CD8b Cell Line

The CD8a molecule, also known as the CD8α chain, is one of the important markers on the surface of immune cells and plays an indispensable role in the immune response of the body. The CD8a molecule belongs to the immunoglobulin superfamily and is encoded by the CD8A gene located on human chromosome 2. It is a glycoprotein that usually exists in the form of a homodimer (CD8αα) and can also form a heterodimer (CD8αβ) with the CD8β chain. The CD8a molecule consists of three parts: the extracellular region, the transmembrane region, and the intracellular region.
CD8B (CD8 Subunit Beta) is a Protein Coding gene. Diseases associated with CD8B include Chromophobe Renal Cell Carcinoma and Uterine Corpus Cancer.

Specifications

Catalog Number	KC-5499
Cell Line Name	CHOK1-CD8a-CD8b Cell Line
Host Cell Line	CHOK1
Description	Stable CHOK1 clone expressing exogenous CD8a-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-CD8a-CD8b cell line was generated using a lentiviral vector expressing the CD8a-CD8b sequence.

Characterization

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

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

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

Figure 4: Characterization of CD8b overexpression in the CHOK1-CD8a-CD8b stable clone using PCR sequence.

Cell Resuscitation

1. Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 10μg/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 (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. Wang L, He Y, Wang P, Lou H, Liu H, Sha W. Single-cell transcriptome sequencing reveals altered peripheral blood immune cells in patients with severe tuberculosis. Eur J Med Res. 2024 Aug 29;29(1):434. doi:10.1186/s40001-024-01991-5. PMID: 39198909; PMCID: PMC11360321.
2.Yang YL, Zhou C, Chen Q, Shen SZ, Li JD, Wang XL, Wang PR. YAP1/Piezo1 involve in the dynamic changes of lymphatic vessels in UVR-induced photoaging progress to squamous cell carcinoma. J Transl Med. 2023 Nov 16;21(1):820. doi: 10.1186/s12967-023-04458-z. PMID: 37974224; PMCID: PMC10655279.
3.Kioussis D, Ellmeier W. Chromatin and CD4, CD8A and CD8B gene expression during thymic differentiation. Nat Rev Immunol. 2002 Dec;2(12):909-19. doi: 10.1038/nri952. Erratum in: Nat Rev Immunol. 2003 Jan;3(1):85. PMID: 12461564.