The K562-Fluc-Puro/HuBCMA-Neo cell line stably expresses firefly luciferase and human BCMA. The cells are designed for in vitro and in vivo studies of BCMA. Parental K562-Fluc-Puro cells can be used as a negative control.

Parental K562-Fluc-Puro cells (Imanis #CL171) cells were transduced with a human BCMA-encoding lentivirus. A high HuBCMA 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 K562 cell line was licensed and purchased directly from ATCC*.

Replication Competent Lentivirus (RCL) Test (including a test report) is available for this cell line at an added cost. Contact us to learn more.

*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 Activity

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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.

BCMA Expression

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K562-Fluc-Puro/HuBCMA-Neo cells were stained with an anti-HuBCMA (blue) or isotype control (grey) antibody and analyzed by flow cytometry.

Surface Receptor Profiling

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K562-Fluc-Puro/HuBCMA-Neo cells were stained with an anti-HuCD20 (A), anti-CD19 (B), anti-HuGPRC5D (C), or anti-HuCD38 (D) antibody and analyzed by flow cytometry.

CAR-T Cell Activation Assay

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Jurkat T cells expressing a BCMA-CAR and GFP were generated by lentivector transduction. Tranduced or untransduced parental Jurkat were incubated in a 1:1 ratio with the indicated K562 cell lines and the following day flow cytometry was performed. The CD3+ Jurkat cells were analyzed for BCMACAR-GFP expression and CD69 expression, which is upregulated in activated T cells.

Complete Growth Medium: Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 15% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin.

For long-term maintenance (more than two to three weeks) addition of 6 µg/mL of puromycin and 1 mg/mL of G418 to the media is recommended to maintain high Fluc and HuBCMA expression.

These cells should be grown in the indicated medium and subcultured as needed to maintain a density between 5 x 10⁵ and 2 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.