Species: Human

Cell type: Colorectal carcinoma

Transgenes: Firefly luciferase (Fluc) with neomycin resistance (Neo) for selection with G418 and near-infrared fluorescent protein (iRFP; ex/em = 690/713) with puromycin resistance (Puro) for selection with puromycin.

Media: DMEM, 10% FBS, 1% Pen/Strep, 0.5 mg/mL G418, 1 μg/mL puromycin

Description: HCT116-Fluc-Neo/iRFP-Puro is a polyclonal population of the human colorectal carcinoma cell line HCT116 (ATCC® CCL-247™) transduced with LV-Fluc-P2A-Neo (LV011) encoding 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 LV-iRFP-P2A-Puro (LV032) encoding nearinfrared 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 HCT116-Fluc-Neo/iRFP-Puro cell line has been tested for mycoplasma contamination and is certified mycoplasma free.

Cell Line Authentication: HCT116 cell line was authenticated are certified free of interspecies cross-contamination by short tandem repeat (STR) profiling with 9 STR loci including CSF1PO, D13S317, D16S539, D5S818, D7S820, TH01, TPOX, vWA and sex chromosome marker Amelogenin.

Recommended uses:

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

In vivo: This human colorectal cell line grows as xenografts in immunocompromised mice. Growth of these tumors can be monitored in vivo using noninvasive bioluminescent imaging with D-luciferin substrate or optical imaging for GFP fluorescence.

Note: Please ensure that your optical imaging system has the appropriate filter sets for imaging iRFP (iRFP; ex/em = 690/713)

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

Flow Cytometry for iRFP: HCT116-Fluc-Neo/iRFP-Puro (red) or control (HCT116; 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 of a subcutaneous HCT116-Fluc-Puro tumor in an athymic mouse

10⁷ HCT116-Fluc-Puro cells (Imanis Life catalog #CL008) 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 days 7 and 16 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#4772) is shown.