For a description of why compensation is necessary, the underlying concepts behind compensation, and some of the pitfalls of improper compensation, see the "Compensation: A Perspective" by Mario Roederer, Ph.D.
FlowJo computes a compensation matrix on control samples much the way you would manually set the compensation. However, software compensation has been shown to be more accurate and reliable than user defined compensation (compensation "by eye"). This is particularly true in experiments using multiple parameters off a single laser (For example: FITC, PE, PerCP and PE-Cy7 off the blue or APC, Alexa700 and APC-Cy7 off the red).
*Please be aware that when using the FCS2.0 data standard, acquisition defined compensation is 'written into' the data file and if samples are over compensated, there is no recourse. If you must use the FCS2.0 data file standard, we recommend that either no acquisition compensation matrix is defined or that samples are under compensated. Therefore, any corrections to the compensation matrix can be made in FlowJo.
**Acquisition compensation matrices defined with the FSC3.0 data standard can be removed and replaced with separate compensation matrices created in FlowJo.
In general, you will create gates on positive and negative populations for each single stain control, and tell FlowJo to calculate the compensation matrix based on these stains. Alternatively, FlowJo's Compensation Wizard can automate this process for you, generating these gates and the matrix in a couple of steps.
Once created, matrices are saved with the workspace. Matrices can be removed, but also can be edited. In addition, matrices can be exported as a matrix file to be loaded into other Workspaces. FlowJo also has the ability to read the compensation matrix created by some other software programs.
There is no limit to the number of matrices that can be created for a given Workspace, and FlowJo gives you the ability to apply a particular matrix to a particular Group, illustrating another way that Groups can be used to help organize your analysis. This is performed by selecting File->Add Matrix to Current Group in the Compensation Window. If you perform Add Matrix to Current Group while holding the shift key down, the matrix will also be associated with the group. New additions to the group will have the matrix applied automatically.
The use of the Compensation Script Tool within the FlowJo allows you to utilize Groups and Keyword combinations to automatically compensate new, identically stained data. To repeatedly perform the same analysis on similar data see Templates.
FlowJo can also transform the display of compensated data so that populations no longer are displayed squished against the axis. This transformation does not alter the data in any way, it simply changes the scale of the axis so that you can view negative numbers and therefore the entire population of cells as a whole cluster. Please visit the display transformation web pages for more information.
You may also click on the topics below to get more help on:
- The Compensation window
- Using the Compensation Wizard
- The compensation matrix file
- Changing/editing the compensation matrix manually
- Display Transformation (for those pesky cells that get squished against the axis!)
- FMO vs. Compensation
- Using the Compensation Scripting Tool (v.7.2.x)
- A Compensation Tutorial (v7.2.x)
Download a Compensation Workspace with Demo Data to try out this platform.
Download the Compensation Tech Note to print a short step-by-step PDF guide.
