What is “Stain Index” and how do I calculate it with FCS Express?
What is the Stain Index?
The Stain Index is a method to help determine the relative intensity for a fluorochrome on a given instrument and is calculated for a fluorescent parameter by subtracting the Median of the negative population from the Median of the positive population, then dividing that quantity by the variance of the negative population, multiplied by 2.
Stain Index = (Median of Positive - Median of Negative) / (SD of Negative * 2)
By determining the relative brightness of a series of fluorochromes, flow cytometry users can simplify the process by which they design an experiment and use the Stain Index metric to match fluorochromes with a higher Stain Index to antigens that are of a lower density or a lower affinity, thus maximizing resolution sensitivity.
Calculating the Stain Index in FCS Express
With FCS Express, you can easily create your own Stain Index to help optimize your experiment for the best possible results. View our example Stain Index data in FCS Express using the free reader, a demonstration download, or your current FCS Express license. You can also view the layout as a PDF.
Resolution sensitivity (the ability to resolve a dim positive signal from background) depends upon the difference between positive and background peak means and the spread of the background peak. The stain index is a metric that captures both of these factors.
Stain index is easily calculated in FCS Express by using integrated spreadsheets. Simply drag and drop statistics from your plots or tables to enter the positive and negative median values in spreadsheet cells a as well as the negative population standard deviation. Once complete, your vales for Stain Index will update in real time as gates or data change in your analysis. View an example PDF of stain index analysis.
The Universal Antibody Titration Template in FCS Express provides users with an easy to use analysis template to evaluate and assess antibody titrations. Open the layout and walk through the Startup Script or follow instructions provided within the layout Standard Operating Procedures. After loading your titration data, adjust parameters, and modify or replace the scatter, negative staining, and positive staining gates as needed. Use the custom tokens to manually enter Titration Antibody and Concentration information and FCS Express will automatically name your batch processing output/report files and populate your spreadsheet for your regression plots.
With a few user actions, the Universal Antibody Titration Layout auto-generates the following regression plots: classic Stain Index, 84th Percentile Stain Index (Separation Index), Signal to noise ratio, and a comparison of the classic Stain Index and Separation Index. The optimal antibody concentration to use is auto-calculated by FCS Express in a spreadsheet using 90% of the vMax from the Stain Index, Separation Index, Signal to noise ratio regression plots. A second spreadsheet contains all the essential antibody titration statistics and is ready for export. A single click on the Run icon from the Batch tab, batch processing will promptly export your titration report files. Titration analysis is simple in FCS Express!
5 point, 6 point, and 7 point Universal Antibody Titration templates are available here:
View the full PDF instructions for step by step guidance on how to use the Universal Antibody Titration Template.
Stain index may also be calculated using the stain index function from the Custom Tokens dialog. Simply drag and drop statistics from your plots or tables to enter the positive and negative median values for the formula as well as the negative population standard deviation. Once complete, the custom token for Stain Index will update in real time as gates or data change in your analysis.
FCS Express 7 now has a Percentile function that allows you to use a defined Percentile within a stain index metric.
The short webinar below describes the theory behind the Stain Index metric and how to use FCS Express to quickly calculate and report Stain Index.
FCS Express on Mac
Upgrading FCS Express