I have been trying to make a drug resistant (Doxorubicin) cell line from HL-60 cells (Suspension culture) but some how I am not able to regrow the drug treated cells even after IC-50 treatment. Can anybody help in this regard?
Did you run a dilution test? You may have to start with low concentrations to keep it sublethal first. Once you get cell growth, try to step up concentration. You may find that survival can be due to lack of entry / reduced rate of entry into the cell.
I have been doing this on a panel of breast cancer cell lines for the last 18 months and have achieved resistance to between 32nM and 140nM depending on the cell line.
Start by splitting your cells into three flasks so they are each about 20% confluence. Then add doxorubicin. Start low. I began with 1nM, 3nM and 5nM. Culture them as you would normally, but include doxorubicin each time you split the cells. When one or more of the three are growing at the same rate as your parental line (by cell counting) freeze it down (as a safety point) and split again into 3 flasks. Keep 1 at the same concentration and use 2 higher doses on the other two flasks. The one maintained at the starting point is your backup in case you increase the dose by too much in the other 2 flasks.
Run a dose increasing MTT on your parental and resistant line to confirm change in IC50.
Bear in mind -
1) Keep a parental line growing at the same time. Length of time in culture and passage number will also have an affect on cell behaviour. Ideally you should have passage number matched parental cells as your controls.
2) Do you want to gradually change gene expression in the entire population or do you want to replicate a crisis in the cell population and select for pre-existing chemo-resistant clones. This will dictate how much you increase the dose by at each round.
3) Are there any other cell lines you may wish to examine in the future - making them resistant at the same time as the HL60's would save you a lot of time later.
4) Do you have the time - it may take anywhere from 2 months to 2 years depending on the cells.
5) HL60's reistant to doxorubicin already exist, would it be quicker to get in touch with the researchers who generated these and get them to send you some?
Good luck.
And also remember your IC50 only refers to the dose required to kill 50% over the length of time you ran the assay for (5 days?). In reality these cells will be growing forever so this value is not very useful to start your experiments. If you can increase your dox dose to the parental IC95 dose then that will be acceptable for publication.
- Badges
- Science topic