KC-4613

293T-SIRPA-KO-1C1 Cell Line

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Background of 293T-SIRPA-KO-1C1 Cell Line

The protein product of SIRPA, signal regulatory protein α1 (SIRPα), also known as CD172a or SHPS-1, is a multifunctional transmembrane glycoprotein.It is a transmembrane protein expressed primarily in macrophages and dendritic cells. SIRPα contains an extracellular region with three immunoglobulin superfamily (IgSF) domains, including a NH2-terminal ligand binding V-domain. It can be activated by CD47 binding, activating a “do not eat me” signal that enables tumor cells to evade immune clearance.

Specifications

Catalog Number	KC-4613
Cell Line Name	293T-SIRPA-KO-1C1 Cell Line
Host Cell Line	293T
Description	Stable 293T clone with human SIRPA gene knockout, No.1C1
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	DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10% FBS
Selection Marker	NA
Morphology	epithelial
Subculture	Split saturated culture 1:5-1:8 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

Cell Line Generation

293T-SIRPA-KO-1C1 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of 293T-SIRPA-KO-1C1 cell line stable clone using PCR sequencing.

Figure 2: Characterization of 293T-SIRPA-KO-1C1 cell line stable clone using RT-PCR sequencing.

Cell Resuscitation

1. Prewarm culture medium (DMEM + 10% FBS)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 1:5-1:8 every 2-3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

1. Prepare the freezing medium (70% DMEM + 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.Zhou Z, Chen MM, Luo Y, Mojumdar K, Peng X, Chen H, Kumar SV, Akbani R, Lu Y, Liang H. Tumor-intrinsic SIRPA promotes sensitivity to checkpoint inhibition immunotherapy in melanoma. Cancer Cell. 2022 Nov 14;40(11):1324-1340.e8. doi: 10.1016/j.ccell.2022.10.012. Epub 2022 Nov 3. PMID: 36332624; PMCID: PMC9669221.
2.Wang P, Song Y, Li H, Zhuang J, Shen X, Yang W, Mi R, Lu Y, Yang B, Ma M, Shen H. SIRPA enhances osteosarcoma metastasis by stabilizing SP1 and promoting SLC7A3-mediated arginine uptake. Cancer Lett. 2023 Nov 1;576:216412. doi: 10.1016/j.canlet.2023.216412. Epub 2023 Sep 26. PMID: 37769797.
3.Logtenberg MEW, Scheeren FA, Schumacher TN. The CD47-SIRPα Immune Checkpoint. Immunity. 2020 May 19;52(5):742-752. doi: 10.1016/j.immuni.2020.04.011. PMID: 32433947; PMCID: PMC7340539.