4T1-Fluc-Neo/eGFP-Puro

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.

eGFP Expression

4T1-Fluc-Neo/eGFP-Puro (green) or control (4T1-mNIS-Puro; grey) cells were fixed with paraformaldehyde and analyzed by flow cytometry (20,000 events).

In Vivo Imaging

4T1-Fluc-Neo cells (Imanis Life catalog #CL020) 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 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.

Complete Growth Medium: RPMI supplemented with 10% FBS, 1X Penicillin/Streptomycin, 0.1 mg/mL G418, and 2 µg/mL puromycin.

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.

4T1 cells frequently clump during growth. When confluent sections of cells become large (e.g. fill an entire field of view at 10X) the cells should be passaged to minimize cell death. For healthy growing 4T1 cells, this usually occurs when the cells reach 80-90% confluency overall.

These cells are suitable for in vitro and in vivo experimentation.

4T1 cells form primary tumors that depending on the route of implantation, can metastasize to the lung, liver, lymph nodes, and brain post implantation into syngenic BALB/c mice.¹

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 LTR². 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:
¹Pulask and Ostrand-Rosenberg. Cancer Res 1998. 58:1486-1493.
²Miyoshi et al. J Virol 1998. 72:8150-8157.