The Nalm6-Fluc-Puro/CD19-KO/HuCD20-Neo (high) cell line stably expresses firefly luciferase and high levels of human CD20, but does not express CD19. To generate this cell line, Nalm6-Fluc-Puro/CD19-KO cells (Imanis #CL180) were transduced with a human CD20-encoding lentivirus. A high HuCD20 expressing population was generated by selection using a methylcellulose based semi-solid medium.

This cell line has been tested for mycoplasma contamination and is certified mycoplasma free.

The parental Nalm6 cell line was licensed and purchased directly from ATCC*.

*The ATCC trademark and trade name and any and all ATCC catalog numbers are trademarks of the American Type Culture Collection.

Morphology

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Phase cell photo taken at 200x magnification

Luciferase Expression

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The indicated number of cells were placed in wells of a 96-well plate. After the addition of 15 mg/mL d-luciferin, bioluminescence was immediately read using a microplate reader.

CD20 Expression

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Nalm6-Fluc-Puro/CD19-KO/HuCD20-Neo (high) cells were stained with an anti-CD20 (blue) or isotype control (grey) antibody and analyzed by flow cytometry.

CD19 Expression

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Nalm6-Fluc-Puro/CD19-KO/HuCD20-Neo (high) cells were stained with an anti-CD19 (blue) or isotype control (grey) antibody and analyzed by flow cytometry.

Surface Receptor Profiling

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Nalm6-Fluc-Puro/CD19-KO/HuCD20-Neo (high) cells (blue) were stained with anti-BCMA (A), anti-GPRC5D (B), anti-CD38 (C), or isotype control (grey; all panels) antibody and analyzed by flow cytometry.

Complete Growth Medium: RPMI-1640 supplemented with 15% fetal bovine serum (FBS), 10 mM HEPES, and 1% Penicillin/Streptomycin.

For long-term maintenance (more than two to three weeks) addition of 1 µg/mL of puromycin and 1 mg/mL G418 can be added to the media.

These cells should be grown in the indicated medium and subcultured as needed to maintain a density between 7 x 10⁵ and 3 x 10⁶ cells/mL. The cells can be passaged by dilution in fresh medium, with occasional passaging using centrifugation to limit the amount of debris in cultures.

These cells are suitable for in vitro and in vivo experimentation. The Fluc transgene facilitates in vivo noninvasive bioluminescent imaging of implanted cells and quantitation of cells in vitro.

These cells were generated via lentiviral vector transduction. The lentiviral vectors used for transduction were self-inactivating (SIN) vectors in which the viral enhancer and promoter have been deleted. Transcription inactivation of the LTR in the SIN provirus increases biosafety by preventing mobilization by replication competent viruses and enables regulated expression of the genes from the internal promoters without cis-acting effects of the LTR¹. Nevertheless, all work with these cells should be performed under biosafety-level 2 (BSL2) conditions by trained personnel. Institutional requirements may permit handling of these cells under BSL1 conditions if certain criteria are met.

1. Miyoshi, H et al. (1998). Development of a self-inactivating lentivirus vector. Journal of Virology 72: 8150-8157.