Hello, dear scientist, I want to use mtt assay to find ic50 of my substance. The substance hadn't tested on the cell line that I choose (PC12). I don't know how to prepare the stock solution.
Dear Elham,
You should choose from as least concentration as possible. Because at higher concentrations, any ways your compound may have general toxicity. You have to first have decide, whether you want to check at nano molar or micro molar range .Then fix the test concentration range (For example: 0, 0.5, 1, 1.5, 2...: this is because you do not know the IC50) along with a positive and a negative control. Based on the concentration range, you have to prepare main stock and working stocks for your experimental triplicates. And check the solubility of your compound, in which solvent it is dissolved completely. Check whether that solvent has any effect on your cell line and your test compound.
Kanugala Sirisha Thanks for your attention, dear Kanugala Sirisha.
You have to take into account that if you want a substance to have a therapeutic benefit clinically acceptable, it has to be at least micromolar range, no more than 10 uM. Usually, approved drugs have around 10 nM. But this is i assume just a preliminary kind of assay, so such a small value wouldn't be expected at this point. Usually, when i prepare stock solutions, I dissolve them above the minimum solubility. DMSO works most of the cases, but check the instructions from your purchase. Make the stock solution in mili molar range. Then, based on the concentrations you want to apply, make the appropriate dilutions to prepare working stock solutions in micro molar range or lower. If oyu have no data on your substance's toxicity, I owuld suggest that you use a wide range of concentration at first, to get a rough estimate on safe window for cell testing. I would do something like that (500nM, 1uM, 50 uM, 200 uM, 500 um). Then, you can fix it to a better range, closing in in the values that you will be investigating
To prepare the stock solution you need to consider several things:
1. Solubility of the substance in different solvents (e.g. water, etahon, DMSO)
2. Toxicity of the solvent in your cell model. This one is related to point 2 since the toxicity of the solvent will be determined in many cases by its final concentration.
3. Sensitivity of your cell line to similar substances. Maybe the specific substance that you are testing has few or no data in this particular cell line. But try searching for the same substance in a different cell model or vice versa, for you to have an idea of the concentration range you will like to use.
4. Expected final concentration of your substance in the cultured cells. Remember that increasing the concentration of your substance, will also increase the concentration of your vehicle. Increasing the chances of having a cytotoxic effect by your vehicle.
Hope this helps
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