Compensation for FlowJo Version 10
Compensation in flow cytometry is the process of correcting for fluorescence spillover emissions. The detectors, or channels on a given cytometer instrument are designed to detect a specific range of emission. However, the fluorescent probes used in flow cytometry generally have a broader range of emission, and are detected by the instrument across multiple detector channels. Each channel will detect the primary fluorescence, but also secondary fluorescence arising from other fluorochromes emitting in the range of that detector. The secondary fluorescence must removed to get an accurate reading in of the primary flurochrome in that
Single stain Compensation controls must be run for each fluorescent reagent being used in a given multicolor experiment in order to determine the amount of secondary fluorescence contributed from each fluorescent reagent to each channel.
tje stained controls used to compensate in FlowJo should to be transformed/scaled correctly before initiating the compensation wizard for best results with the automatic gating of Size and Negative/Positive subsets.
For version 10, there are three main components to the user interface for Compensation:
- The Compensation group – in the workspace and tied to compensation Preferences
- The Compensation window – used to define a compensation matrix from single stain controls
- The Matrix editor – used to view/adjust matrix numbers with a preview of the changes.
One other document you will likely find useful is:
Applying/Removing – the matrix, a small overview.
When you are ready to proceed to a more complete overview of the new compensation work flow, please see this document.
Here is a collection of external links about Compensation.
All of the pages linked above, also link back here for your convenience.
Questions? Feel free to contact us at flowjo@bd.com