Parental U266B1 cells were transduced with LV-SFFV-Luc2-P2A-Puro (Imanis #LV012) and LV-SFFV-eGFP-P2A-Neo (Imanis #LV067), which encode the firefly luciferase (Fluc; Luc2) and enhanced green fluorescent protein (eGFP) cDNAs, respectively, under the spleen focus-forming virus (SFFV) promoter and linked to antibiotic resistance (puromycin, neomycin) genes via a P2A cleavage peptide. A high Fluc and eGFP expressing population was generated by selection using puromycin and G418 followed by selection with a methylcellulose based semi-solid medium.

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

The parental U266B1 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 and fluorescence cell photos 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 3 mg/mL d-luciferin, bioluminescence was immediately read using a plate reader. The total bioluminescence (RLU) was plotted as a function of cell number.

EmGFP Expression

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U266B1-Fluc-Puro/eGFP-Neo (green) or parental U266B1 (grey) cells were fixed with paraformaldehyde and analyzed by flow cytometry.

Surface Receptor Profiling

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U266B1-Fluc-Puro/eGFP-Neo (green) cells were stained with isotype control antibody (grey) or antibody specific to human BCMA (A), CD19 (B), CD20 (C), GPRC5D (D), or CD38 (E) and analyzed by flow cytometry.

Complete Growth Medium: ATCC-formulated RPMI-1640 supplemented with 15% fetal bovine serum (FBS), 1% Penicillin/Streptomycin, 3 μg/mL puromycin, and 1 mg/mL G418.

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. eGFP is not recommended for in-life imaging, but can be used for post-mortem confirmatory assays. Both Fluc and eGFP facilitate quantitation of cells in vitro.

These cells were generated via lentiviral vector transduction. The lentiviral vector used for transduction was a self-inactivating (SIN) vector 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.