KC-3285

CHOK1-mouse-CD28 Cell Line

34294

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Background of CHOK1-mouse-CD28 Cell Line

CD28, also known as TP44, is a single-pass type I membrane protein. CD28 is one of the molecules expressed on T cells that provide co-stimulatory signals, which are required for T cell activation. CD28 is the receptor for CD80 and CD86. When activated by Toll-like receptor ligands, the CD80 expression is upregulated in antigen-presenting cells. The CD86 expression on antigen-presenting cells is constitutive. CD28 is the only B7 receptor constitutively expressed on naive T cells.

Specifications

Catalog Number	KC-3285
Cell Line Name	CHOK1-mouse-CD28 Cell Line
Host Cell Line	CHOK1
Description	Stable CHOK1 clone expressing exogenous mouse CD28 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% 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
In Vivo Validation	NA

Cell Line Generation

CHOK1 mouse CD28 cell line was generated using a lentiviral vector expressing the mouse CD28 sequence.

Characterization

Figure 1: Characterization of mouse CD28 overexpression in CHOK1 stable clones using FACS.

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 (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. Evans EJ, Esnouf RM, Manso-Sancho R, Gilbert RJ, James JR, Yu C, et al. (March 2005). Crystal structure of a soluble CD28-Fab complex. Nature Immunology. 6 (3): 271-9. doi:10.1038/ni1170. 2. Expansion of cytotoxic CD8 + CD28-T cells in healthy ageing people, including centenarians. Immunology. 88(4): 501-507. 3. Functional Subsets within Clonally Expanded CD8+ Memory T Cells in Elderly Humans. Clinical Immunology.94 (3): 160-172.

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.