You should first test the sensitivity of your cells through a cell viability assay (e.g.: ATP assay, MTT...).
Then when you found cells sensitive to your drug of interest, you chronically treat them with a concentration of drug for which you see very low viability.
You have to refresh your drug media regularly (period depends on your drug half life).
You proceed like this until emergence of resistance.
You should observe death of most of your cells, with very few cells surviving the process(possible resistant clones).
It can be very long process several months, with high risk of contamination with mold and bacteria since you don't split the cells and keep them in the same dish (you only refresh media)
If your drug is not too experimental you should easily find protocol on the web with the right concentration to use and the period for drug refreshing.
Hope it helps,
You should first test the sensitivity of your cells through a cell viability assay (e.g.: ATP assay, MTT...).
Then when you found cells sensitive to your drug of interest, you chronically treat them with a concentration of drug for which you see very low viability.
You have to refresh your drug media regularly (period depends on your drug half life).
You proceed like this until emergence of resistance.
You should observe death of most of your cells, with very few cells surviving the process(possible resistant clones).
It can be very long process several months, with high risk of contamination with mold and bacteria since you don't split the cells and keep them in the same dish (you only refresh media)
If your drug is not too experimental you should easily find protocol on the web with the right concentration to use and the period for drug refreshing.
Hope it helps,
I use a prolonged drug selection technique to enrich resistant cancer cells in vitro which was based on growing cancer cells in the presence of anti-cancer agent starting from low concentration and continuing with double the amount of previous concentration (until you reach to IC100 or IC200 concentration of the drug for the cell line you are working with). In each step dead cells are eliminated and the new concentration is given to cells in fresh media. This way you eliminate cells that are sensitive to the drug and those which survive are the ones that are resistant to the drug. After enriching this population the media you maintain them should involve a certain concentration of the same drug.
This article can be a good example for what I have described above:
Check out the material method-cell line part where the authors describe how they generated adriamycin resistant breast cancer cells.
Article High levels of transglutaminase expression in doxorubicin-re...
Deniz is also correct.
You can also go with incremental concentration of drugs, however such protocol might allow adaptive response to the drug. So depending on what you want to achieve acute treatment might be preferable.
in my experience, selection of resistant cancer cells from a cell line or a primary culture is feasible through dose-escalating method, i.e., starting from low concentration of the drug, one that would induce cell growth arrest or even cell death, but not massive cell death. After some days (this will depend on your experimental conditions), you will see growth of some cells. At this point you should wash the dead cells and the medium off the dish and add fresh medium with a 50% higher dose of the drug. Again, you will see arrest and death... You repeat this until you reach a concentration that on one hand, let the resistant cells grow and on the other hand, kill all of the parental, untreated cells. This approach is similar to the one that Deniz Cansen described. I haven't tried the acute treatment as suggested by Ludovic, but I agree it could be worth testing it also. I hope it helps. Best, Mariano
Dear Sudip,
- Badges
- Science method