JoVE Encyclopedie van Experimenten
Biologische Technieken
0 weergaven • 4:33 min. • July 8th, 2025
During cancer metastasis, tumor cells from a primary tumor travel through the circulatory system, extravasate to distant organs, and establish secondary tumors.
To evaluate lung metastasis in a breast cancer mouse model that has been injected with 4T1 cells, highly metastatic breast cancer cells, prepare a cell suspension, including 4T1 cells, isolated from the harvested lungs. Centrifuge the suspension and resuspend the cells in media containing 6-thioguanine, 6-TG - a guanine analog. Seed the cell suspension in a culture plate.
6TG enters normal cells where specific transferase enzyme converts it to 6-TG nucleotide, a toxic nucleotide. These cytotoxic nucleotides get incorporated into DNA, causing DNA damage, and selectively eliminate cells from culture. 4T1 cells have decreased ability to form toxic nucleotides, facilitating their survival and adherence to the plate. Depending on the proliferative ability of cells, they form colonies.
Treat the cells with methanol. Methanol permeabilizes the cells and denatures the cellular proteins. Incubate the cells with methylene blue.
The positively-charged dye enters the cell, interacts with the negatively-charged DNA, intercalating into s
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