A dose response assay is an experiment designed to measure the effect of different concentrations of a drug or other treatment on a biological system. This can be used to establish the optimal concentration for use in future experiments, but also allows you to determine if there is any effect at all and if so what that effect might be. To do this, we need to perform some simple tests on cells in culture (in vitro). If you want to test whether cytokine produced by these cells increases when treated with different concentrations of a drug, then it's important that the number of cells per well does not exceed the growth rate: too many cells will overgrow before secreting cytokine can be measured.
If you are using serum, make sure that the concentration is not too high. As a rule of thumb, use an equal volume of serum to cells. For example, if you are using 1x106 cells per well, it is safe to add 1 ml of 20% FBS (v/v) to each well. On the other hand, if your cell density is low, you may add more FBS than what we recommend above and still maintain a reasonable response within a reasonable time frame (such as 24 hours). You should be careful when adding more than 20% FBS because cytokines may begin to be released from viable tissue in the medium and this will have an effect on your results!
Similarly with plates containing media instead of serum or complete medium: always use more media than expected if possible since this will result in greater numbers being tested at once without affecting our ability to detect differences between these doses/concentrations with statistical significance between them (see below).
You can also use different ratio of cell plating to make sure that your cells are not too crowded. For example, if you want to measure the dose response effect of TNFα in a low amount of supernatant and need to detect for relatively small differences in cell signaling activity, then use 1:1 or 1:2 ratio of cells per well instead of using 1:10 (or more). In this case, it is best if you have enough cytokine in your supernatant so that there will be enough agonist ligand to activate NF-κB-dependent gene expression. However, if you have only low amount of cytokine in supernatant and need to detect subtle changes in NF-κB activation by this low amount of ligand that may cause little difference between groups but still significant biologically speaking (as we discussed above), then it is better to choose higher ratios since they allow more precise detection.
This is very critical when you have low amount of cytokine in supernatant. For example, the concentration of IL-6 in your supernatant may only be at 1 ng/mL and you want to know what is the dose response curve. You can use more cells (i.e., increase cell density) and get an idea about the dose response curve by plotting log(IL-6) vs Log [cells]/ml.
A common mistake when performing dose response assays is not accounting for the growth rate of your cells. In order to do this, you'll need to know two things:
Make sure you do not use to many cells per well, as it will overgrow before you can measure cytokine secreted. A good rule of thumb is no more than 100,000 cells per well. Also, make sure that you have enough volume of media in each well so that the cells can grow out to confluency before being stimulated with your antigen/antibody mixture. This will ensure that each sample has equal growth conditions, and therefore equal amounts of cytokine secreted for comparison.
You should use a standard curve to determine the amount of cytokine secreted by your cell line (see below). First add an appropriate amount of control proteins (IgG or IgM) at various concentrations ranging from 0-10 ug/ml (or whatever range you are using) into wells containing non-treated cells. Then add 50 μl of this solution onto wells containing treated cells and incubate overnight at 37 C° in 5% CO2 incubator.
Use a standard curve if possible when measuring cytokine secretion levels in one group versus another
The dose response assay is one of the most popularly used biochemical assays for analyzing drug binding to receptors. It can be used to determine the affinity of a drug for its receptor and also help predict its effectiveness in vivo. A good example of this would be aspirin, which has an extremely high affinity for COX-1 and COX-2 but doesn’t induce inflammation because it does not achieve 100% occupancy of these receptors.
You will need:
As you increase the amount of drug, you will notice a parallel increase in binding with the receptor.
This is because the drug is occupying more receptors.
The more receptors that are occupied, the more binding there will be.
When you plot these values using Microsoft Excel and choose Linear Regression Analysis, you will get a plot showing the slope and y-intercept. The y-intercept represents the amount of binding of your receptor with the drug while the slope represents the amount of drug required to achieve 50% occupancy of the receptors.
The dose response assay is a useful tool to determine affinity constants between drugs and receptors. The affinity constant indicates how tightly a drug binds to its receptor. The higher the affinity, the more likely it is that an organism will respond to treatment with that drug.
We hope this helped you to understand how to do a dose response assay. If you have any questions or comments please leave them in the comment section below.