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	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
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.