KC-1720

CHOK1-SIRPAv2-Cell-Line

22681

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Background of CHOK1-SIRPAv2-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. The cell surface receptor signal-regulatory protein-α (SIRP-α) is expressed on inflammatory cells and suppresses phagocytosis. 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 that protects cells from phagocytosis by binding and activating its receptor SIPRA on macrophages. Diseases associated with SIRPA include Intellectual Developmental Disorder, X-Linked 50 and Keratopathy.

Specifications

Catalog Number	KC-1720
Cell Line Name	CHOK1-SIRPAv2-Cell-Line
Host Cell Line	CHOK1
Description	Stable CHOK1 cell line expressing exogenous human SIRPAv2 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 24 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

CHOK1-SIRPAv2 Cell Line was generated using a lentiviral vector expressing the human SIRPAv2 sequence.

Characterization

Figure 1: Characterization of SIPRAv2 overexpression in the CHO-K1 SIPRAv2 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (RPMI1640 supplemented with 10% FBS and 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 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% RPMI1640 + 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

Tomiyama T, Itoh S, Iseda N, Toshida K, Kosai-Fujimoto Y, Tomino T, Kurihara T, Nagao Y, Morita K, Harada N, Liu YC, Ozaki D, Kohashi K, Oda Y, Mori M, Yoshizumi T. Clinical Significance of Signal Regulatory Protein Alpha (SIRPα) Expression in Hepatocellular Carcinoma. Ann Surg Oncol. 2023 Jun;30(6):3378-3389. doi: 10.1245/s10434-022-13058-y. Epub 2023 Jan 15. PMID: 36641515.
Garcia-Sanchez C, Casillas-Abundis MA, Pinelli DF, Tambur AR, Hod-Dvorai R. Impact of SIRPα polymorphism on transplant outcomes in HLA-identical living donor kidney transplantation. Clin Transplant. 2021 Sep;35(9):e14406. doi: 10.1111/ctr.14406. Epub 2021 Jul 11. PMID: 34180101.
Londino JD, Gulick D, Isenberg JS, Mallampalli RK. Cleavage of Signal Regulatory Protein α (SIRPα) Enhances Inflammatory Signaling. J Biol Chem. 2015 Dec 25;290(52):31113-25. doi: 10.1074/jbc.M115.682914. Epub 2015 Nov 3. PMID: 26534964; PMCID: PMC4692235.