LL/2-Fluc-Puro is a polyclonal population of the Lewis lung carcinoma cell line LL/2 (ATCC® CRL-1642™). To achieve stable reporter expression in the polyclonal population, parental LL/2 cells were transduced with LV-Fluc-P2A-Puro (LV012) and selected using puromycin. LV-Fluc-P2A-Puro encodes the firefly luciferase (Fluc) cDNA linked to the puromycin resistance gene (Puro) via a P2A cleavage peptide under the spleen focus-forming virus (SFFV) promoter.

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

The parental LL/2 cell line has been authenticated and certified free of interspecies cross contamination by short tandem repeat (STR) profiling with 9 STR loci.

Due to the immunogenicity of the reporter genes in this cell line, we recommend using immunocompromised mice for in vivo studies.

Replication Competent Lentivirus (RCL) Test (including a test report) is available for this cell line at an added cost.  Contact us to learn more.

Morphology

Low and high density cell morphology (200x)

Luciferase Expression

 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.

In Vivo Imaging

1e7  LL/2-Fluc-Puro cells 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#6867) is shown.

Complete Growth Medium: DMEM supplemented with 10% FBS, 1X Penicillin/Streptomycin, and 2 µg/mL puromycin.

The addition of puromycin to the complete growth medium maintains high reporter expression over continued passage of the cells. It is highly recommended, especially if the cells undergo multiple passages prior to being used for studies.

These cells should be grown in the indicated medium and passaged when they reach confluency. For routine passaging, cells are recommended to be split at a 1:10 ratio every 3-4 days.

These cells are suitable for in vitro and in vivo experimentation. LL/2 cells form tumors and lung metastases post implantation into syngenic C57BL mice.^1,2

The Fluc transgene facilitates in vivo noninvasive bioluminescent imaging of implanted cells.

The cells can be amplified in vitro and used to generate additional frozen stocks. Cryopreservation of low passage stocks is recommended. Frozen stocks should be preserved in a designated cryopreservation medium.

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 LTR³. 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.

References:
¹Bertram et al. Cancer Letters. 1980. 11:63-73.
²Takeda et al. (2015) Blood 118: 464-472.
³Miyoshi et al. J Virol 1998. 72:8150-8157.