KC-1694

CHOK1-cyno-SIRPA-Cell-Line

22631

Home » CHOK1-cyno-SIRPA-Cell-Line

Background of CHOK1-cyno-SIRPA-Cell-Line

Signal regulatory protein alpha (SIPR alpha), also named CD172a, is a regulatory membrane glycoprotein that belongs to the SIRP/SHPS(CD172) family of the immunoglobulin superfamily. SIRPa is expressed mainly on myeloid cells, including macrophages, neutrophils, dendritic and Langerhans cells, and also stem cells or neurons. SIRPa acts as inhibitory receptor through recognizing with a broadly expressed transmembrane protein CD47, this interaction also calls “don’t eat me” signal, and negatively regulates phagocytosis of innate immune cells.

Specifications

Catalog Number	KC-1694
Cell Line Name	CHOK1-cyno-SIRPA-Cell-Line
Host Cell Line	Chinese hamster ovary CHO-K1 cell line
Description	CHOK1 cell line stable expressing exogenous crab-eating macaque SIRPa 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 SIRPa cell line was generated using lentiviral vector expressing SIRPa sequence of crab eating monkey.

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

Kharitonenkov, A. et al. A family of proteins that inhibit signalling through tyrosine kinase receptors. Nature vol. 386 181ÿ186 (1997).
Voets, E. et al. Functional characterization of the selective pan-allele anti-SIRPϫ antibody ADU-1805 that blocks the SIRPϫ-CD47 innate immune checkpoint. J. Immunother. Cancer 7, 1ÿ15 (2019).