I am looking to find a protocol suitable for a learning environment, where cells will be fed an anti-cancer drug and then stained for actin and tubulin to check the cytoskeleton irregularities that arise. Wanted more specifically to know which drugs (nacodazone, colchicine, taxol, etc) would have a greater affect on the cytoskeleton, how long to incubate the cells for, and the concentration. The cells used could even be fibroblasts instead of cancer cells, just as long as the difference in the cytoskeleton was evident enough to demonstrate to the students.
Phalloidin, tubulin tracker should help. A good epithelial cell type would reflect the changes more readily.
Hi Raquel,
You can employ Tubulin polymerization assay for your purpose. You can also use confocal laser scanning microscopy (CLSM) after staining cells with respective fluorescent antibodies. Following links may be useful for you.
http://www.cytoskeleton.com/pdf-storage/datasheets/bk006p.pdf
https://www.cellsignal.com/browse/?Ntk=Products&N=4294956287&Ntt=tubulin+
https://www.cellsignal.com/products/antibody-conjugates/vimentin-d21h3-xp-rabbit-mab-alexa-fluor-647-conjugate/9856?N=4294956287&Ntt=vimentin&fromPage=plp
The most typical experiment for assessing cytoskeleton is to detect actin polymerization assay. You can find the detailed methods in my paper:
https://www.researchgate.net/publication/294918637_Hedgehog_inhibitors_selectively_target_cell_migration_and_adhesion_of_mantle_cell_lymphoma_in_bone_marrow_microenvironment
Hope it would help you!
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