It should be by hit and trial method or based on published literature.

You must search the pubmed and check whether someone tested similar compound to yours. If there is no data regarding your compound, then I would use wide range of concetrations - begining from nM to uM, for example 10 increasing concentrations. Then it is trial and error.

I also would suggest to try to find similar nanocomposites tested by other researchers, first. If your material is very expensive, I would try 3 different concentrations at first, from very high to very low (like 100 times different). If not, you could try to test 6 or 8 different concentrations, 10 times different from each other. On the second round, I would make double dilutions around your estimated EC50 value in the first experiment.

From my experinece, MTT test is usually not very suitable for some types of nanoparticles, so also choose the right method. Cell titer Glo is very good, though it is quite expensive.

Thank you all for your input.

Vilma Petrikaite In case of MTT assay, if the nanoparticles are insoluble in cell culture media, then how do we deal with this situation. Because if we add suspension form of nanoparticles to cells, that will not reflect the actual concentration of nanoparticles we want to add.

Yes, it is quite tricky.

When you dissolve chemical compounds or soluble NPs, they are distributed in the medium. When you add insoluble particles, they usually used to sediment on the cells. In this case the concentration is just a relative dimension, the surface area of the well and amount of cells are even more important. Sometimes NPs are used to attach to the bottom of well, and the cells do not have space to attach after dividing, thus the toxicity of NPs could also come from physical contact, not only due to the loaded material toxicity, etc. And before measurement absorbance of many reagents used for cytotoxicity evaluation, it is very important to wash out NPs with PBS several time, unless you already proved that NPs do not have any influence on reagent absorbance/fluorescence/luminescence values.