This question is for those who have already used the BD Cycletestet Plus: I'm using this kit for cell cycle arrest analysis on cancer cells subjected to treatment with natural compounds. Some researchers agree synchronizing cells, while others don't. I wonder who is correct.
So, about the second question: I've used IC50 and IC20 values based on MTT assay in other experiments (LDH assay and Annexin/PI flow cytometry) . Should I use the same concentrations?
Neither is "correct". You need to consider the factors in your experiment. For example, could depriving the cells of serum further sensitize them to drug and would that be acceptable for this study? Also, if this is a single cell line and all of your plates were seeded from the same stock at the same time and cultured under the same conditions then it may be ok to assume that their cell cycles are already somewhat synchronous. If they are different cell lines then you will likely want to synchronize. Just be sure to document exactly what methodology you used for publication. If you have the time and resources it doesn't hurt to perform the experiment both ways (*this will increase your confidence in your results if this is an important concern for you).
Regarding concentration: You should select a range of concentrations. Maybe use your IC20 and IC50 in addition to a very low and a very high concentration. The more concentrations you use the more information you will obtain about how your drugs affect the cell cycle.
If your purpose is to observe the effect of the drugs I don't believe that you have to synchronize your cells, just be sure that they are at a density that allows for exponential growth when harvesting. Try to use a drug concentration that will reflect practical clinical drug levels.
Thanks! These informations will be very important for my project.
Synchronize cells with 24hr serum starvation before drug treatment. Drug concentration depends on cells and drug. You can start with low and move towards high concentration.
Thanks for everybody who have helped me! I've done the cell cycle test without synchronizing cells, just using negative and positive controls to compare.
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