U266B1-Fluc-Puro/BCMA-KO is a clonal population of the multiple myeloma cell line U266B1 that has been engineered to stably express firefly luciferase and lack BCMA expression.
The U266B1-Fluc-Puro/BCMA-KO cell line was generated by transfecting Imanis U266B1-Fluc-Puro (CL173) cells with Cas9-gRNA ribonucleoprotein complexes targeting human BCMA. Individual cell clones were generated using a methylcellulose based semi-solid medium and screened for BCMA expression by flow cytometry. Sequencing of the targeted BCMA region confirmed genetic knock-out in the selected cell clone.
This cell line has been tested for mycoplasma and no mycoplasma has been detected. The parental U266B1 cell line was authenticated by short tandem repeat (STR) profiling.
Low and high density cell photos (200x).
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 TECAN microplate reader.
U266B1-Fluc-Puro/BCMA-KO cells were stained with an anti-BCMA (blue) or isotype control (grey) antibody and analyzed by flow cytometry. (Note: cells were grown in the presence of g-secretase inhibitor to stabilize any surface BCMA expression.)
U266B1-Fluc-Puro/BCMA-KO cells were stained with an anti-HuCD19 (A), anti-HuCD20 (B), anti-HuCD38 (C), or anti-HuGPRC5D (D) antibody and analyzed by flow cytometry.
Imanis cell lines U266B1-Fluc-Puro/BCMA-KO, U266B1-Fluc-Puro/BCMA (mid), and U266B1-Fluc-Puro were stained with anti-human BCMA antibody (blue) or isotype control (grey) and analyzed by flow cytometry.
Complete Growth Medium: ATCC-formulated RPMI-1640 supplemented with 15% fetal bovine serum (FBS), and 1X Penicillin/Streptomycin.
The addition of 3 µg/mL puromycin to the complete growth medium is recommended if maintaining the cells in culture for more than two weeks.
Grow cells in the indicated medium, subculturing as needed to maintain a density between 1×105 and 1.5×106 cells/mL. Passage cells by dilution in fresh medium with regular complete media changes. For optimal growth and viability, a complete media change should be performed every 3-4 days even if the cells are not ready to passage.
These cells are suitable for in vitro and in vivo experimentation.
The luciferase transgene facilitates noninvasive bioluminescent imaging of implanted cells in vivo and cell-based assays in vitro.
U266B1 cells are tumorigenic in immunocompromised mice, exhibiting selectivity for the bone marrow compartment.
These cells can be amplified to generate additional frozen stocks and cryopreservation of low passage stocks in a designated cryopreservation medium is recommended.
These cells were generated by transduction with a self-inactivating Risk Group 3 (RG3) lentiviral vector. All work should be performed under appropriate biosafety conditions per institutional guidelines.
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