- Frozen (CL110) $ 1,800
Cell type: Melanoma
Transgenes: Firefly luciferase (Fluc) with neomycin resistance (Neo) for selection with G418 and near-infrared fluorescent protein (iRFP) with puromycin resistance (Puro) for selection with puromycin
Media: DMEM, 10% FBS, 1% Pen/Strep, 0.8mg/mL G418, 1 µg/mL puromycin
Description: B16F10-Fluc-Neo/iRFP-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-iRFP-P2A-Puro (LV032) encoding infrared fluorescent protein (iRFP) cDNA under the SFFV promoter and the puromycin resistance gene (Puro) via a P2A cleavage peptide.
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).
Mycoplasma Testing: The B16F10-Fluc-Neo/iRFP-Puro cell line has been tested for mycoplasma contamination and is certified mycoplasma free.
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.
In vitro: This is a high Fluc/iRFP expressing cell line suitable for use as a positive control cell line in bioluminescence assays and fluorescence assays to verify Fluc or iRFP 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 iRFP fluorescence.
Note: Please ensure your optical imaging machine has the correct excitation and emission filters for iRFP detection.
Morphology: Low- and high-density cell morphology (200x)
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.
Flow cytometry for iRFP: B16F10-Fluc-Neo/iRFP-Puro (red) or control (B16F10; grey) cells were fixed with paraformaldehyde and analyzed by flow cytometry (20,000 events).
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.