KC-1611

293T-CD47-KO-1D3-Cell-Line

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Background of 293T-CD47-KO-1D3-Cell-Line

CD47, also named as integrin associated protein (IAP) is a transmembrane protein belonging to the immunoglobulin superfamily, which acts as a “don’t eta me” signal to macrophage after binding with its ligand signal-regulatory protein alpha (SIRPa), and is potential therapeutic target in some cancer and treatment of pulmonary fibrosis.

Specifications

Catalog Number	KC-1611
Cell Line Name	293T-CD47-KO-1D3-Cell-Line
Host Cell Line	Human HEK293T cell line
Description	293T cell line stable clone with CD47 gene knockout
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% DMEM+20% FBS+10% DMSO
Propagation Medium	DMEM+10%FBS
Morphology	Epithelial
Subculture	Split saturated culture 1:3-1:6 every 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

293T-CD47-KO cell line was generated using CRISPR method.

Characterization

Cell Resuscitation

Prewarm culture medium (DMEM + 10% FBS)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 1:3-1:6 every 3 days; seed out at about 1-3 × 10⁵ cells/mL.

Cell Freezing

Prepare the freezing medium (70% DMEM + 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

Willingham, Stephen B, Jens-Peter Volkmer, Andrew J Gentles, Debashis Sahoo, Piero Dalerba, Siddhartha S Mitra, Jian Wang, et al. 2012. ¡ùThe CD47-Signal Regulatory Protein Alpha (SIRPa) Interaction Is a Therapeutic Target for Human Solid Tumors..¡ì Proceedings of the National Academy of Sciences of the United States of America 109 (17). National Acad Sciences: 6662Ã¿67.
Vonderheide, Robert H. 2015. ¡ùCD47 Blockade as Another Immune Checkpoint Therapy for Cancer.¡ì Nature Medicine 21 (10). Nature Publishing Group: 1122Ã¿23. doi:10.1038/nm.3965.