|FlowJo gives you the ability to compensate your data. This may be necessary in cases where the compensation was inappropriately set during sample collection (although if the sample was over-compensated, then there is no recourse). Also, there may be cases where the instrument is not capable of compensating between certain channels (for instance, to correct for the spillover between fluorescein and Cy5PE).
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 (you need to be connected to a network to view this site). The remainder of the documentation below is NOT meant to teach you about compensation, simply how to use FlowJo to perform that function.
FlowJo computes the compensation matrix on control samples much the way you would manually set the compensation. To do this, you will select gates on positive and negative populations for each of these stain, and tell FlowJo to calculate the compensation matrix based on these stains.
Thus, you must collect samples that are singly-stained (as well as unstained) for each of the fluorescences that require compensation. Ideally, you would use a stain that only labels a portion of the sample population, so that you have an unstained set of cells in each tube. It is important to remember that the negative population and positive population must share the same autofluorescence characteristics for compensation to be valid (i.e., don't use monocytes as a negative control for a lymphocyte stain; if you are compensating a stain on fibroblasts, use an unstained fibroblast control).
Steps involved in compensation by FlowJo:
- Define positive and negative gates for each fluorescence channel requiring compensation
- Open the compensation window; drag the positive and negative populations into the appropriate boxes
- Compute and save the matrix
- Apply the matrix to the appropriate samples
If you need to generate another compensation matrix for other samples in the experiment, you can just repeat steps 2 through 4 as needed.
You may also view an example of the entire compensation process, as a tutorial.
Once you have defined a compensation matrix, it is saved with the workspace. You may subsequently apply that compensation matrix to other samples in the same workspace by selecting it from the menu. To use that matrix in other workspaces, save the matrix to a file ("Edit/Save Matrix") and load the matrix file into the new workspace ("Load matrix"). Note that a compensation matrix is generally valid only for samples collected during a single collection run! However, you can also edit the matrix using the "Edit/Save Matrix" option.
Any sample that has been compensated is marked with a blue bar next to the sample name in the workspace window. Compensated samples have new parameters added to their list: for each fluorescence channel to be compensated, a new parameter is created. The parameter name is bracketed with "<>": e.g., when FITC and PE are compensated against each other, two new parameters named "<FITC>" and "<PE>" will be created. Remember to select these new parameters in the graph or statistics windows!
A new feature in FlowJo Version 4.3 transforms 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:
Download a Compensation Workspace with Demo Data to try out this platform.
Download the Compensation Tech Note to print a short (four page) step-by-step guide.