KC-3032

CHOK1-cyno-CD28-Cell-Line

Stable CHOK1 cell line expressing exogenous cyno CD28 gene

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Background of CHOK1-cyno-CD28-Cell-Line

CD28 is a membrane protein of B7 receptor family and mainly expressed on T cells. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins, and provide co-stimulatory signals for T cell activation and survival.

Specifications

Catalog Number	KC-3032
Cell Line Name	CHOK1-cyno-CD28-Cell-Line
Description	Stable CHOK1 cell line expressing exogenous cyno 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-like
Subculture	Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-2 × 10⁵ cells/mL
Incubation	37 °C with 5% CO₂
Storage	Liquid nitrogen immediately upon receiving
Doubling Time	Approximately 24 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

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

Characterization

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS + 10µg/mL Puromycin) in a 37°C water bath.
Thaw the frozen vial in a 37°C water bath for 1-2 minutes.
Transfer the vial into biosafety cabinet, and wipe the surface with 70% ethanol.
Unscrew the top of the vial and transfer the cell suspension gently into a sterile centrifuge tube containing 9.0mL complete culture medium.
Spin at ~ 125 × g for 5-7 minutes at room temperature, and discard the supernatant without disturbing the pellet.
Resuspend cell pellet with the appropriate volume of complete medium and transfer the cell suspension into a T25 culture flask.
Incubate the flask at 37°C, 5% CO₂ incubator.
Split saturated culture 1:4-1:8 every 2-3 days; seed out at about 1-2 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI-1640 + 20% FBS + 10% DMSO) fresh immediately before use.
Keep the freezing medium on ice and label cryovials.
Transfer cells to a sterile, conical centrifuge tube, and count the cells.
Centrifuge the cells at 250×g for 5 minutes at room temperature and carefully aspirate off the medium.
Resuspend the cells at a density of at least 3×10⁶ cells/mL in chilled freezing medium.
Aliquot 1 mL of the cell suspension into each cryovial.
Freeze cells in the CoolCell freezing container overnight in a -80°C freezer.
Transfer vials to liquid nitrogen for long-term storage

References

Linsley PS, Ledbetter JA (1993). "The role of the CD28 receptor during T cell responses to antigen". Annu. Rev. Immunol. 11: 191ÿ212.
Lenschow DJ, Walunas TL, Bluestone JA (1996). "CD28/B7 system of T cell costimulation". Annu. Rev. Immunol. 14: 233ÿ58.