I will treat our suspension cell line with bioactive compound to determine the effect of proliferation for 6 days. What is the common protocol for this experiment? When I will change the medium, should I add fresh compound in it? How am I sure about the concentration which I will add to the medium?
The medium should be changed every 48 hours. The medium is supplemented with fresh compound. Some researchers prefer to change half the medium (with medium containing fresh compound) every 48 hours.
I agree to Syed. I also know lab that exchange 90% of the medium regularly and keep 10% of the used medium (it contains growth factors and similiar stuff that the cells secreted). The concentration of compound can be calculated for the volume of fresh medium only or (especially if the compound is known to be instable) for the final volume (fresh and used medium). Make sure that you know the relationship of compound concentration (added freshly) and cell number for each change.
For an idea of starting concentration either search the literature (if it's a known compound) or try different concentrations, differing by factor 2, 5 or 10 and also try extreme (low and high) concentrations. It's a matter of availability and price of the compound of course.
Don't forget control experiments: How is the normal proliferation rate of your cells without any additional thing? How is the proliferation rate with the solvent (e.g. DMSO or whatever your compount is solved in) of your compound only? Does it have an influence? If possible it might improve your work if you can monitor the natural "break-down" of your compound in culture medium under culturing conditions (temperature, CO2). Depending on your compound this might be easily possible with UV spectroscopy or chromatographic procedures.
Based on my experience there is not a single protocol for this type of treatment: kind of compound and cellular cytotypes are variables to consider each experiment. However, in agreement with previous speeches, the medium should be changed every 48 hours; personally, I change the medium completely and, each time, I add fresh compounds. About the concentration which you add to the medium I'm agree with Vivica Grotelueschen: examine the literature or try different concentrations.
Personally, first time that I add a bioactive compound, I use the best efficacy concentration but, subsequently, I use a concentration progressively lower; in this way, I prevent the cytotoxic effect of the compound. I treat my suspension and adherent cell lines with bioactive compound also for 21days: it's very important, for me, to avoid cytotoxic effects.
Best regards
First of all when you do long term survival or colony formation assay, you need to keep low concentration of your compound. Secondly, cells should be treated with compound only once of a definite time period (24-48h depending upon doubling time of cells). Change the medium with fresh medium without adding your compound and grow for 6-10 days (change the medium after every 48h without adding drug). You can find the detail protocol in this particular paper..
http://www.impactjournals.com/oncotarget/index.php?journal=oncotarget&page=article&op=view&path%5B%5D=6134&path%5B%5D=15205
I agree with Stefano; the medium containing the compound should be refreshed every 48h. The same operation must be performed for the control conditions
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