LL/2-eGFP-Puro is a polyclonal population of the Lewis lung carcinoma cell line LL/2 (ATCC® CRL-1642™).
LL/2-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-eGFP-PGK-Puro (LV031) and selected using puromycin. LV-eGFP-PGK-Puro encodes the enhanced green fluorescent protein (eGFP) cDNA under the spleen focus-forming virus (SFFV) promoter and the puromycin resistance gene (Puro) under the mouse phosphoglycerate kinase (PGK) 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.
A C57Bl/6 mouse was implanted with 5 x 105 LL/2-eGFP-Puro (CL051) cells in the right hind flank. Tumor growth was monitored over time using calipers. Fluorescence imaging was performed on Day 16 using an IVIS Spectrum.
Low and high density cell morphology (200x)
LL/2-eGFP-Puro cells (green) or control (LL/2, grey) cells were fixed with paraformaldehyde and analyzed by flow cytometry (20,000 events).
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
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. (2011) Blood 118: 464-472.
3Miyoshi et al. J Virol 1998. 72:8150-8157.
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