I'm a student in computational drug design, but I'm going to move to the bench soon to test some compounds we've designed. I've been reading the methods sections of some papers describing novel drugs, and I have a couple of questions.
1) How is the range of concentrations to test determined? For example, one report described a concentration range of 30 uM to 1 nM. Are these numbers chosen arbitrarily, or are they based on something?
2) I sometimes see the concentrations written like this: "75 uL of 2x drug solution (3-fold dilutions, 10 uM maximum concentration)". I understand the 3-fold dilution part, but what does the 2x refer to? 2x the IC50? 2x some other concentration?
1) If the compound is new and you don't know its EC50 against any cell line, I would recommend at first to do a preliminary study using a wide range of concentrations, eg. from 100 uM to 1 nM (use 10x dilutions). Of course, before doing experiment, check the solubility of your tested compound, as you have to be sure that in all those concentrations it is dissolved. Then you will find an approx. EC50, and you may narrow concentration window so, that your EC50 would be somewhere in the middle. Let's say, during the preliminary experiment EC50 was about 2 uM. Then you could make 2X dilutions starting from 10 uM (10; 5; 2.5; 1.25; 0.625; 0.3125 uM).
2) I agree that such description is confusing. It should be good to know the context in which it was mentioned. I guess, somebody wrote 2x drug solution just because usually people make 2x more concentrated solutions for proliferation assays, as later they dilute it twice with the cell culture medium when they put it into the plates with growing cells. Maybe somebody had 75 uL of medium in each well and added 75 uL of tested drug.
A perfect answer. Just in case 10X dilutions term is confusing: You have 100 ul of cell suspension per well of 96 well plate. To test in wide range of concentrations, you need to add 100 ul of 2X compound solution to each. The 2X solutions will have 200uM, 20uM, 2uM, 0.2uM of your compound to make 200ul suspension of cells mixed with compound in each well at final concentration of compound of 100uM, 10uM, 1 uM, etc, etc...
Dear you may find useful information in links below:
https://pdfs.semanticscholar.org/cc49/b6a1cc7c521d7964248d68e79c4658dd84df.pdf
https://www.news-medical.net/.../Quantitative-Measurement-of-Cancer-Cell-Proliferati...
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Materials Science
- Materials
- Inorganic Materials
- Ceramics
- Glass