Preparation of Reporter-Expressing Cancer Cells for In Vivo Imaging

Begin with adherent human breast cancer cells.
Add recombinant adenoviruses carrying a luciferase reporter gene under the control of a TGF-β/Smad-responsive promoter and incubate.
The adenoviruses bind to the cells, enter and deliver the DNA.
Later, endogenous TGF-β/Smad3 signaling activates the Smad3 transcription factor, which binds to the promoter and initiates luciferase expression.
Remove the media to eliminate extracellular viruses and wash with a buffer.
Add trypsin-EDTA, gently shake, and incubate to detach the cells from the surface.
Add a neutralizing solution to terminate the trypsin action and collect the suspension.
Centrifuge to separate the cells. Remove the supernatant containing trypsin-EDTA.
Resuspend the cells in a fresh medium and transfer to a tube.
Add a basement membrane matrix.
Store the luciferase-expressing cells at a low temperature for further analysis.
48 hours before tumor implantation, seed approximately 2.5 times ten to the sixth MDA-MB-231 cells into ten 175 square centimeter flasks.

After 24 hours in culture, add 300 microliters of adenovirus CAGA12-luciferase into each flask before returning the cells to the incubator for another 24 hours. On the day of impla