This article covers the details of bell-shaped dose response plots, a concentration depended curve, to draw the efficacy of therapeutic agents across chosen range of concentrations for the molecules. We believe that you've created protocols before, or if you have not, you can start with this introductory tutorial.
Bell-shaped Dose-response calculation entails the following steps-
- Creating Readout for input data with or without normalization
- Creating Readout to calculate intercept values and plot curve
- Data calculations for different intercepts
- Editing, Setting and Saving the curve
Once you've created a new protocol that has, at least, a name and a project, you will need to enter your readout definitions. Generally, numeric readouts (like the Y-axis Measurement and Concentrations of tested samples) will be used to plot a Bell-shaped Dose Response curve, so these numerical readouts will need to be added to your Protocol first.
Creating Readout for input data with or without normalization
Create a Readout to contain the concentration values:
Create a Readout to contain the Y-axis measurement values:
If the data has been collected using negative or positive or both controls then choose the normalization at this step and define your controls either at protocol default plate layout or plate specific layout.
Creating a Readout to calculate intercept values and plot curve
To complete the Dose-Response calculation, fill in the form working from top to bottom.
- start by selecting the "Plot" data type,
- then select the X-axis and Y-axis from the drop-down list of existing readouts,
- optionally constrain your fit parameters if required,
- optionally select a custom Inactive Range, and
- select which data calculation you want (ie, EC50, EC90, or custom etc)
- Select intercept side for both
The following equation has been used to generate Bell-shaped curves and determine the height or dip of the peak, plateaus, slopes and centers:
Plateaus: If the curve goes up first, then plateau1 is on the left. If the curve goes down first, plateau1 is on the right. The plateaus are the limit of Y-axis as you approach ± infinity in X-axis.
Peak is the plateau level in the middle of the curve.
Slopes: You might consider constraining these to equal 1.0 (stimulation) and -1 (inhibition).
Centers: The centers of the two Hill Equations that are being added together.
Data calculations for different intercepts
Select pre-defined end-point calculations, or create your own custom calculations for both sides of the curves by entering a name, specifying the desired intersection and side of intercept. EC90 and EC99 are the concentrations that give 90% stimulatory and inhibitory effects in the same units as X in the example here.
Don't forget to set the display format for data calculations to the desired number of decimal places or significant figures. This works just like in Excel - the underlying calculations are performed on the full values, but the displayed result is rounded per your display settings.
Now you're done. Don't forget to click "add/update calculation" at the bottom of the form.
Here's what the bell-shaped curve looks like:
Editing, Setting and Saving the curve
By clicking at the curve, you can enter into another tab where you can edit the plot, outliers and override this curve.
Individual outliers can be omitted by clicking the point if there is any in the dataset. You can zoom the curve by selecting the area using the mouse. Different colour, size and shape can be selected for each curve.
Setting tab at top left corner allows to hide grid or intercepts. Also you can add the title of the plot in the free text here. If required then there is a possibility to switch to summary curve.
At the statistics tab you can add arbitrary line and whiskers.
Each curve can be adjusted for chosen fit parameters and it will override the parameters that has been chosen at protocol level.
Once you've defined the settings then you can save the curve either as PNG or PDF image from the top banner of the curve.