Hi Everyone,
I have a work to test IC50 of many kinds of chemicals for lymphoma cell lines. I would like to set up 10 concentration gradient on 96 well plates, but I'm not sure how to decide the highest concentration of these chemicals.
Some one suggested me to start from 5uM and following by 2-fold dilution, because too high concentration may amplify noise from toxicity of chemicals, compared to the effect for specific target. However, I saw some chemicals used in previous articles showed effect with much higher concentration. So, how about the start concentration you set up usually? Thanks!
The choice of concentrations should be based on what you know about the system you are working with. Do you expect the inhibitors to be relatively weak or potent, or some of each? How soluble do you expect the compounds to be? If you don't know what to expect, then you might want to cover a very wide range by using 3- or 4-fold serial dilutions. If you prefer 2-fold serial dilutions (as I do), then it might be better to make 2 separate series, a high-concentration one and a low-concentration one. If you want to cover a wide range of potencies while still using 2-fold serial dilutions, you can just make more dilutions, such as 20 instead of 10, using 2 rows instead of 1.
When I used to do high-throughput screen hit follow-up IC50 measurements, it was common to prepare 2-fold serial dilutions with a top tested concentration of 100 µM or 200 µM. If the IC50 was lower than the lowest tested concentration, the IC50 could be repeated with a lower concentration range.
You have to review what is said about your compound in the literature, then test several dilutions/concentrations of the compound. As in the calculation of the IC50, you will need to convert your concentrations (x axis) into logarithms, it can be interesting to test logarithmically increasing concentration. For example, as you are expecting (according to previous research I guess) that the IC50 would be about 5 µM, one approach could be to test following concentrations: 0.1 µM, 0.3 µM, 1 µM, 3 µM, 10 µM, 30 µM, 100 µM, and 300 µM. Then you will measure the cell viability at these concentrations, and you can calculate the IC50 by plotting these values on a graph showing log concentrations on the x-axis and cell viability on the y-axis. Ideally, you should obtain a "S" shaped curve and the IC50 concentration should be located an inflection point of the curve. You can use a software (GraphPad Prism, Excel, …) to easily compute this.
As already mentioned, first of all they need information about the substance, i.e. in which range the toxicity takes place. Then you can perform a 1:2 dilution series as a screening. For the range 10% living cells and at least 30% dead cells we then create dose factors which result from the 2 to 12 root 10. You then get a table that covers a decade at a time. This is a good way to narrow down the IC50 range. For the 8 root the following sequence of numbers would result: 10; 13,3; 17,8; 23,7; 31,6; 42,2; 56,2: 75; 100.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques
- Analytical Chemistry
- pH