KC-0249

293T-human-CD47-Cell-Line

46437

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Background of 293T-human-CD47-Cell-Line

CD47, also known as integrin-associated protein (IAP), is a transmembrane protein that belongs to the immunoglobulin superfamily. It functions as a “don’t eat me” signal to macrophages by binding to its ligand, signal regulatory protein alpha (SIRPα). CD47 is considered a potential therapeutic target in certain cancers and in the treatment of pulmonary fibrosis.

Specifications

Catalog Number	KC-0249
Cell Line Name	293T-human-CD47-Cell-Line
Host Cell Line	293T
Description	Stable HEK293T clone expressing exogenous human CD47 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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 0.5μg/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 30 hours
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

293T human-CD47 cell line was generated using a lentiviral vector expressing the human-CD47 sequence.

Characterization

Figure 1: Characterization of human-CD47 overexpression in the 293T human-CD47 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 0.5μg/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% 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,et al. 2012.“The CD47-Signal Regulatory Protein Alpha (SIRPa) Interaction Is a Therapeutic Target for Human Solid Tumors.” Proceedings of the National Academy of Sciences of the United States of America 109(17). National Acad Sciences: 6662–67. doi:10.1073/pnas.1121623109.
Vonderheide, Robert H. 2015. “CD47 Blockade as Another Immune Checkpoint Therapy for Cancer.” Nature Medicine 21(10). Nature Publishing Group: 1122–23. doi:10.1038/nm.3965..