KC-3434

293T-cyno-CD20-Cell-Line

27003

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Background of 293T-cyno-CD20-Cell-Line

CD20, also known as MS4A1, CVID5, encodes a member of the membrane-spanning 4A gene family, members of which are characterized by common structural features and similar intron/exon splice boundaries and display unique expression patterns among hematopoietic cells and nonlymphoid tissues. This gene encodes a B-lymphocyte surface molecule which plays a role in the development and differentiation of B-cells into plasma cells. Researches have shown that anti-CD20 monoclonal antibodies selectively deplete CD20+ B and CD20+ T cells and efficiently suppress inflammatory disease activity and constitute immunotherapies for the treatment of B cell malignancies and autoimmune diseases.

Specifications

Catalog Number	KC-3434
Cell Line Name	293T-cyno-CD20-Cell-Line
Host Cell Line	293T
Description	Stable 293T clone expressing exogenous cyno-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% DMEM + 20% FBS + 10% DMSO
Propagation Medium	DMEM + 10% FBS + 1μg/mL Puromycin
Selection Marker	Puromycin
Morphology	Epithelial
Subculture	Split saturated culture 1:4-1:8 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-cyno-CD20-cell-line was generated using a lentiviral vector expressing the cyno-CD20 sequence.

Characterization

Figure 1: Characterization of cyno-CD20 overexpression in the 293T-cyno-CD20 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 1:4-1:8 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

de Sèze J, Maillart E, Gueguen A, Laplaud DA, Michel L, Thouvenot E, Zephir H, Zimmer L, Biotti D, Liblau R. Anti-CD20 therapies in multiple sclerosis: From pathology to the clinic. Front Immunol. 2023 Mar 23;14:1004795.
Kumar A, Planchais C, Fronzes R, Mouquet H, Reyes N. Binding mechanisms of therapeutic antibodies to human CD20. Science. 2020 Aug 14;369(6505):793-799.
Ly S, Nedosekin D, Wong HK. Review of an Anti-CD20 Monoclonal Antibody for the Treatment of Autoimmune Diseases of the Skin. Am J Clin Dermatol. 2023 Mar;24(2):247-273.