U266B1-Fluc-Puro/BCMA-KO
- Frozen / Standard (CL184) $ 3,500
$3500
Species: Human
Cell Type: Multiple Myeloma
Transgene: Firefly luciferase (Fluc; Luc2)
Selection Gene: Puromycin resistance (Puro)
Knock-out: B-cell maturation antigen (BCMA)
Description:
The U266B1-Fluc-Puro/BCMA-KO cell line stably expresses firefly luciferase and does not express BCMA. To generate this cell line, U266B1-Fluc-Puro (Imanis #CL173) cells were transfected 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 contamination and is certified mycoplasma free.
This cell line is also available for purchase as a pair with the U266B1-Fluc-Puro (BCMA positive) cell line, for a discounted rate (link).
Characterization:
Morphology
Phase cell photo taken at 200x magnification
Luciferase Expression
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
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.)
Surface Receptor Profiling
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.
Growth Conditions
Complete Growth Medium: ATCC-formulated RPMI-1640 supplemented with 15% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin.
For long-term maintenance (more than two to three weeks) addition of 3 µg/mL of puromycin to the media is recommended.
These cells should be grown in the indicated medium and subcultured as needed to maintain a density between 1 x 105 and 1.5 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. For optimal growth and viability, do a media change every 3-4 days even if the cells are not ready to passage.
Usage Information
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.
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Miyoshi, H et al. (1998). Development of a self-inactivating lentivirus vector. Journal of Virology 72: 8150-8157.
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