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Compensation Overview

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).

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:

  1. Define positive and negative gates for each fluorescence channel requiring compensation
  2. Open the Define Matrix tab; drag the positive and negative populations into the appropriate boxes
  3. Compute and save the matrix
  4. 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.

FlowJo's compensation tool and its derived parameter tool are combined in one window, shown below. With a population selected in the Workspace, select Compensation from the Tools menu. Follow the links below for a more detailed description.

  1. Clicking here activates the compensation process. If you have more than one matrix in the tool, choose the one you want to work with from this list. The Define Matrix tab lets you drop positive and negative gated subpopulations populations from the Workspace to define the matrix values for this parameter.
  2. The Edit Matrix tab lets you change the matrix numbers manually and also save and load matrices.
  3. The Assign Tubes tab lets you drag and drop uncompensated populations (or groups of them) to a matrix in order to apply the matrix to the samples.
  4. The list of a population's parameters appears here. Drag the subset formed by gating the population that is negative for a parameter to the box marked Negative.
  5. Drag the subpopulation gated as positive for a parameter to this box.
  6. Simplify the process by dragging the parent of the two gated populations to the AutoAssign Box. Or control-select all parent populations after gating and drag them here.
  7. Clicking here switches to the Derived Parameter tools.

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 Matrix tab, Save to File button) and load the matrix file into the new workspace (Edit Matrix tab, Load from File button). 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 Matrix tab.

Any sample that has been compensated is marked with a 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 labeled with "comp-": e.g., when FITC and PE are compensated against each other, two new parameters named Comp-FITC and Comp-PE will be created. Remember to select these new parameters in the graph or statistics windows when you want to view compensated data.

Name your matrix at any time by selecting Untitled-1 in the box at the top and typing in a name of your choice. The new name will appear in the Compensation Matrices list at the left of the Compensation tool.

FlowJo can 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:

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.
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