LL/2-Fluc-Neo/eGFP-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-Neo (LV011) and LV-eGFP-PGK-Puro (LV031) and selected using G418 and puromycin. LV-Fluc-P2A-Neo encodes the firefly luciferase (Fluc) cDNA linked to the neomycin resistance gene (Neo) via a P2A cleavage peptide under the spleen focus-forming virus (SFFV) promoter. LV-eGFP-PGK-Puro encodes the enhanced green fluorescent protein (eGFP) cDNA under the SFFV promoter and the puromycin resistance gene (Puro) under the phosphoglycerate kinase promoter (PGK).

*The ATCC trademark and trade name and any and all ATCC catalog numbers are trademarks of the American Type Culture Collection.

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)

eGFP Expression

LL/2-Fluc-Neo/eGFP-Puro (green) or control (grey) cells were fixed with paraformaldehyde and analyzed by flow cytometry (20,000 events).

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

A C57Bl/6 mouse was implanted with 5 x 10⁵ LL/2-Fluc-Neo/eGFP-Puro (CL073) cells through the tail vein. Bioluminescence imaging was performed on the indicated days using an IVIS Spectrum. Fluorescence imaging of the extracted lungs was performed upon autopsy using the IVIS Spectrum.

Complete Growth Medium: DMEM supplemented with 10% FBS, 1X Penicillin/Streptomycin, 2 µg/mL puromycin, and 1.25 mg/mL G418.

The addition of G418 and puromycin to the complete growth medium maintains high dual 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. eGFP is not recommended for whole animal in-live imaging. Rather, samples can be collected postmortem for analysis by conventional fluorescence microscopy or flow cytometry.

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.