KC-1400

293T-CD24 Cell Line

22065

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Background of 293T-CD24 Cell Line

CD24, also known as heat stable antigen or small-cell lung carcinoma cluster 4 antigen, is a heavily glycosylated glycosylphosphatidylinositol-anchored surface protein and is expressed on B cells, T cells, keratinocytes, and myofiber synaptic nuclei and is upregulated in a wide variety of cancers. CD24 is a potent anti-phagocytic, ‘don’t eat me’ signal that is capable of directly protecting cancer cells from attack by Siglec-10-expressing macrophages. Siglec-10 as an innate immune checkpoint that is essential for mediating anti-tumor immunity and provides evidence for the therapeutic potential of CD24 blockade, with promise for the treatment of ovarian and breast cancers.

Specifications

Catalog Number	KC-1400
Cell Line Name	293T-CD24 Cell Line
Host Cell Line	293T
Description	Stable 293T cell line expressing exogenous CD24 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 + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:5 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-CD24 cell line was generated using a lentiviral vector expressing the CD24 sequence.

Characterization

Figure : Characterization of CD24 overexpression in the 293T-CD24 stable clone using FACS.

Cell Resuscitation

Prewarm culture medium (DMEM supplemented with 10% FBS and 1μg/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 1:4-1:5 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

K Pirruccello, S. J. & LeBien, T. W. The human B cell-associated antigen CD24 is a single chain sialoglycoprotein. J. Immunol. 136, 3779–3784 (1986).
Chen, G. Y., Brown, N. K., Zheng, P. & Liu, Y. Siglec-G/10 in self–nonself discrimination of innate and adaptive immunity. Glycobiology 24, 800–806.
Amira A. Barkal, et al. CD24 signaling through macrophage Siglec-10 is a target for cancer immunotherapy. Nature research.2019.