The stain index measures the relative brightness of various fluorochromes on a given instrument. This value can be used to compare and rank fluorochrome brightness and is a useful tool to determine optimal titer for antibody titration, which is essential to generate high-quality cytometry data. Using too little antibody can result in incomplete labeling, whereas using too much antibody can lead to increased background fluorescence and binding of the antibody to low-affinity sites (false positive) while making experiments more expensive.
The stain index is defined as the ratio of the separation between the median fluorescence index of positive and negative population divided by two times the standard deviation of the negative population.
Figure1: Stain index. The stain index is the ratio of the separation between the median fluorescence index of positive population (green) and the negative population (black), divided by two times the standard deviation of the negative population.
How to create the Stain index chart
In the Cytobank platform, you can use the Stain Index chart to determine the best antibody concentrations for your titration. The optimal dilution can be determined based on the Stain Index chart to achieve a bright signal with the least background noises. To create a Stain Index chart:
1) Upload the titration samples into a new experiment. Make positive and negative gates for the titrated reagent channels using the histogram plots. You may use range gates or split gates. If you use range gates, there have to be gates drawn on the positive and negative region respectively. If you use split gates, just name your gates and the Cytobank platform will calculate the positive and negative portion accordingly.
Adjust the gates to fit all files, as the marker expression may shift due to different titration. Remember to click on Apply next to the plot to apply the gates to the illustrations.
2) Tag your samples with Doses and Reagents dimensions.
Click on Sample tags>Add experiment dimensions, in the next window, click on Tag files with Doses and Reagents.
3) Tag the files based on the dilutions. Below is an example where the antibody was titrated from 2X (2 times the recommended antibody concentration) to 1:32. You may see more about sample tagging here: How to annotate data with sample tags in Cytobank.
(Tag files with Doses(left) and Reagents(right))
4) Once the files are tagged, click on Illustrations in the blue navigation bar on the top and create a new illustration. In the Illustration Editor, configure the Plots and Layout as shown below. Select Line chart as Plot type, Stain index to be on; X-axis group needs to be Doses and Reagents as Table 1. You may rearrange the dilution order by clicking on the file selection to the right of the Doses dimension. Here is more information about the Layout setting.
(Plots and Layout setting for the Stain Index chart)
5) The Stain Index chart can be saved and copied out of the Illustration Editor directly. The Stain Index Statistics table below the chart shows the stain index value of each dilution for that particular reagent.