KC-1322

CT26.WT-human-CD20-Cell-Line

21925

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Background of CT26.WT-human-CD20-Cell-Line

CD20 (MS4A1) is a glycosylated phosphoprotein mainly expressed on the cell surface of all B-cells beginning at the pro-B phase (CD45R+, CD117+) and progressively increasing in concentration until maturity. CD20 is a highly attractive target antigen of several approved monoclonal or bispecific antibodies, which are all active agents in the treatment of all B cell lymphomas, leukemias, and B cell-mediated autoimmune disease.

Specifications

Catalog Number	KC-1322
Cell Line Name	CT26.WT-human-CD20-Cell-Line
Host Cell Line	CT26.WT
Description	Stable CT26.WT cell line expressing exogenous human CD20 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 1:4-1:10 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	NA
Mycoplasma Status	Negative
In Vivo Validation	NA

Cell Line Generation

CT26.WT human CD20 Cell Line was generated using a lentiviral vector expressing the human CD20 sequence.

Characterization

Figure 1: Characterization of human CD20 overexpression in the CT26.WT human CD20 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 1:4-1:10 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

Tedder TF, Streuli M, Schlossman SF, Saito H (January 1988). Isolation and structure of a cDNA encoding the B1 (CD20) cell-surface antigen of human B lymphocytes. Proceedings of the National Academy of Sciences of the United States of America. 85 (1): 208–12