Species: Mouse

Cell type: Melanoma

Transgenes: Firefly luciferase (Fluc) with neomycin resistance (Neo) for selection with G418 and enhanced green fluorescent protein (eGFP) with puromycin resistance (Puro) for selection with puromycin

Media: DMEM, 10% FBS, 1% Pen/Strep, 0.8mg/mL G418, 1 ug/mL puromycin

Description: B16F10-Fluc-Neo/eGFP-Puro is a polyclonal population of the murine melanoma cell line B16F10 (ATCC^® CRL-6475^™) transduced with 1) LV-Fluc-P2A-Neo (LV011) encoding the firefly luciferase (Fluc) cDNA under the spleen focus-forming virus (SFFV) promoter linked to the neomycin resistance gene (Neo) via a P2A cleavage peptide and 2) LV-eGFP-PGK-Puro (LV031) encoding enhanced green fluorescent protein (eGFP) cDNA under the SFFV promoter and the puromycin resistance gene (Puro) under control of the PGK promoter.

The lentiviral vector used is a self-inactivating (SIN) vector in which the viral enhancer and promoter has 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 LTR (Miyoshi et al., J Virol. 1998).

Cell Line Authentication: The parental B16F10 cell line was authenticated and are certified free of interspecies cross-contamination by short tandem repeat (STR) profiling with 27 STR loci.

Recommended uses:

In vitro: This is a high Fluc/eGFP expressing cell line suitable for use as a positive control cell line in bioluminescence assays and fluorescence assays to verify Fluc or GFP expression respectively in your lentiviral transduced cells.

In vivo: B16F10 cells form metastases in the lungs of mice. The in vivo growth of these metastases can be monitored using bioluminescent imaging with D-luciferin substrate or optical imaging for GFP fluorescence.

Note: In-life imaging for GFP fluorescence is not recommended due to high background autofluorescence. Tissues may be harvested post mortem for analysis by conventional microscopy.

Publications that used associated reagents: 

LV-eGFP-PGK-Puro (LV031): Shen et al. Immunovirotherapy with vesicular stomatitis virus and PD-L1 blockade enhances therapeutic outcome in murine acute myeloid leukemia. Blood. 2016. March 17: 127(11): 1449-58. 

Morphology: Low- and high-density cell morphology (200x)

Flow Cytometry for eGFP: B16F10-Fluc-Neo/eGFP-Puro (green) or control (B16-Fluc-Neo; grey) cells were fixed with paraformaldehyde and analyzed by flow cytometry (20,000 events). 

Luciferase Assay: 10⁴, 10⁵, or 10⁶ cells were placed in wells of a 96-well plate and 0.3 mg of d-luciferin was added to the indicated wells. The plate was immediately imaged using a Xenogen IVIS Spectrum.

Bioluminescent images showing growth of a subcutaneous B16F10-Fluc tumor in an athymic mouse

10⁷ B16-Fluc-Puro cells (Imanis Life catalog#CL052) were injected subcutaneously into the right flanks of female NCR athymic mice. Mice were imaged at day 0 on the day of cell implantation and at day 7 using a Perkin Elmer IVIS® Spectrum system, at 10-15 minutes post intraperitoneal injection of D-luciferin at 150 mg/kg. Tumor size was measured using calipers. Data from a representative mouse (ID#6383) is shown.