KC-6695

Ramos-CD19-CD20-KO-luc2 Cell Line

64663

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Background of Ramos-CD19-CD20-KO-luc2 Cell Line

The CD19 and MS4A1 (CD20) genes, located on chromosomes 16p11.2 and 11q12.2 respectively, encode two canonical B-cell lineage-specific surface antigens. CD19 functions as a critical signal transduction component of the B-cell receptor co-receptor complex, thereby modulating B-cell activation, differentiation, and proliferation. CD20, a four-transmembrane domain phosphoprotein, serves as a store-operated calcium channel essential for B-cell cycle progression and humoral immune responses. Together, these molecules are indispensable for normal B-cell development and immunological function.The clinical relevance of CD19 and CD20 extends beyond basic immunology. Their high and stable expression on the surface of most B-cell malignancies has established them as primary targets for immunotherapy, including chimeric antigen receptor (CAR) T-cells and monoclonal antibodies (e.g., Rituximab). However, lineage plasticity and antigen loss represent major resistance mechanisms, often leading to post-therapeutic relapse. Consequently, dual targeting of CD19 and CD20 has emerged as a promising strategy to mitigate antigen escape and improve durable responses, with multiple bispecific CAR-T constructs currently in preclinical and clinical development.

Specifications

Catalog Number	KC-6695
Cell Line Name	Ramos-CD19-CD20-KO-luc2 Cell Line
NCBI/UniProt Accession Number	NM_001770.6, NM_152866.3
Clone Number	2C1
Host Cell Line	Ramos-Luc2
Description	Stable Ramos-Luc2 cell clone with knockout of CD19 and CD20 genes
Quantity	One Vial 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
Selection Marker	NA
Morphology	Lymphoblast
Subculture	Split saturated culture 1:4-1:8 every 1-3 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

Cell Line Generation

Ramos-CD19-CD20-KO-luc2 cell line was generated using the CRISPR method.

Characterization

Figure 1: Characterization of Ramos-CD19-CD20-KO-luc2 cell line stable clone using PCR sequencing.

Figure 2: Characterization of Ramos-CD19-CD20-KO-luc2 cell line stable clone using RT-PCR sequencing.

Figure 3: Characterization of Ramos-CD19-CD20-KO-luc2 cell line stable clone using FACS.

Figure 4: Characterization of the Ramos-CD19-CD20-KO-luc2 cell line stable clone using Bright-Lite Luciferase Assay System in the conditions of different cell numbers.

Cell Resuscitation

Prewarm culture medium (RPMI1640 + 10% FBS) 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 1-3 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

Kordic A, Phillips TJ, Weiss J. The Current State of Bispecific Antibodies and T-Cell Directed Therapy in NHL. Cancers (Basel). 2025 Mar 31;17(7):1192. doi: 10.3390/cancers17071192. PMID: 40227768; PMCID: PMC11988123.
Montagna, E., de Campos, N.S.P., Porto, V.A. et al. CD19 CAR T cells for B cell malignancies: a systematic review and meta-analysis focused on clinical impacts of CAR structural domains, manufacturing conditions, cellular product, doses, patient’s age, and tumor types. BMC Cancer. 2024;24(1):1037. doi: 10.1186/s12885-024-12651-6.PMID: 39174908; PMCID: PMC11340198.

Use License Agreement

Research Use Only.

Not for use in diagnostic procedures or therapeutic applications.

Redistribution of the cell line or its derivatives is prohibited without prior written permission from Kyinno Biotechnology.