Parental RPMI-8226 cells were transduced with LV-SFFV-Luc2-P2A-EmGFP (Imanis #LV050), encoding the firefly luciferase (Fluc; Luc2) cDNA under the spleen focus-forming virus (SFFV) promoter and linked to the emerald green fluorescent protein (EmGFP) cDNA via a P2A cleavage peptide. A high Fluc and eGFP expressing population was generated by selection using a methylcellulose based semi-solid medium. Note that this this clone expresses moderate levels of CD20.

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

The parental RPMI-8226 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

Phase cell photos taken at 200x magnification

Luciferase Activity

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.

Surface Receptor Profiling

RPMI-8226-Fluc-EmGFP (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), and 1% Penicillin/Streptomycin.

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 a complete media change recommended approximately every four days for optimal cell viability.

These cells are suitable for in vitro and in vivo experimentation. The Fluc transgene facilitates in vivo noninvasive bioluminescent imaging of implanted cells. EmGFP is not recommended for in-life imaging, but can be used for post-mortem confirmatory assays. Both Fluc and EmGFP 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.