I am treating my cells (MEF) with three different concentrations (1um, 10um and 20um) of various small molecules (M1, M2,.....,M10). Some of them show dose-independent behavior on cells but others are not following any trend. For example, Less effective at 1um, more at 10um, less at 20um and vice versa. I am wondering that does it happen with some molecules? If yes then what is the reason? Please help me.
Note: These molecules are not toxic.
This behavior is very interesting. Such results could be in the case when your compound is specific to its target at lower concentration and becomes unspecific (it binds to other targets) at higher concentrations. You can prove such action using a knockout cell line that does not have your target. However, sometimes such results can be obtained when you use different concentration of compound solvent for different doses, also when you use to many cells/per well and they can overgrow, etc.
Do you do your experiments in the same conditions? Eg. adding drugs always 24h after cells seeding, keeping the drug always 48h and keeping always the same density of seeding? Those parameters are crucial to obtain repeatable results. Furthermore the time incubatoin with the drug could be to short. Eg. when I treated cells which had the doubling time of 40h with the drug by 24h I observed such unspecific behaviour.
Dear Dr. Vilma, proving dose dependent specificity is a good suggestion. But it would be too difficult to find that where this is bind non-specifically. Is there any easy way to prove it? Second, cell density is not too highh and my wells never got 100% confluent.
Dear Rimple, I think it is not due to target saturation because in that case the effect must be constant at higher concentration too. No fluctuations but in my case I can see random fluctuations.
Dear Dr. Agnieszka, I am doing experiments in the same conditions. I am following the conditions mentioned in the literature with reference to my cell line and drug target. I am using the MEF cell line (doubling time around 36 hrs). Can you give me any suggestions about drug treatment time?
Depends on structure and functional sites of your molecules.They may or may not interact with membrane pore proteins.
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