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The CD20 gene is located in the 11q12 region of human chromosome 11 and encodes a transmembrane phosphoprotein with a molecular weight of approximately 33–37 kDa. It is primarily expressed during the mature stages of B-lymphocyte development (ranging from pre-B cells to pre-plasma cells). This protein consists of four transmembrane domains and two extracellular loops; while its intracellular segment lacks enzymatic activity, it participates in regulating B-cell receptor signaling and calcium influx—specifically by forming homotetramers—thereby playing a pivotal role in the processes of B-cell activation, proliferation, and differentiation. The CD20 protein is specifically expressed within the B-cell lineage (with the exception of plasma cells), rendering it a critical therapeutic target for B-cell malignancies. Furthermore, CD20-targeted therapies have demonstrated potential utility in the treatment of autoimmune diseases (such as rheumatoid arthritis and multiple sclerosis), and the mechanisms governing its expression regulation remain a focal point of current immunological research.

Figure 1. CD20 KO Luc Raji cells are derived from Raji-Luc cells through the stable knockout of human CD20.

Figure 2.  Sanger of Raji Human CD20 KO Luc Cell, chr11-60463104-C (MS4A1:p.L88Sfs*30)    MS4A1(NM_021950.4): c.262del C.


Figure 3. CD20 KO Luc Raji Cells: Luciferase Validation Results. Detect Luciferase assay by Ultra Luciferase Detection Kit RQPH0001.

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed Raji Human CD20 KO Luc Cell complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.