Compensation is used to ensure that the signal in a single detector is coming from the assigned fluorophore and not spillover from another fluorophore in the panel. Proper compensation is necessary to ensure that false positives are not being analyzed. However, if a small panel was designed in such a way that spillover between all flurophores is negligible then compensation is not needed. There are several rules that must be followed to ensure that compensation can be properly calculated:

1. The positive population must be as bright or brighter than the multicolor sample
2. The control should ideally be exactly the same fluorophore as the one in the sample
3. The control should ideally contain a positive and negative population, and these populations must have the same autofluorescence properties
4. Controls should have sufficient events in both positive and negative populations

More information on the best practices for creating compensation controls can be found in the Flow Basics 2.4 video, which discusses all experiment controls.

In order to calculate compensation, there are many options available: automated tools, manual adjustment, software on the cytometer, analysis software. The video below explains the different routes that can be taken and how to choose the best method.

There are several posts on our blog that provide useful tips for compensation. Links to these posts are provided below.