CompensationTraditional Flow Cytometry
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:
- The positive population must be as bright or brighter than the multicolor sample
- The control should ideally be exactly the same fluorophore as the one in the sample
- The control should ideally contain a positive and negative population, and these populations must have the same autofluorescence properties
- 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.
The Right and Wrong Way to Set Up Automated Compensation Tools: How to Achieve Accurate Compensation
When calculating compensation, automated tools are the gold standard. However, people often struggle to get good results from the automated compensation tools and will turn to manual compensation to fix any errors. Why is it difficult to get accurate results...
I have an unpopular opinion: I love compensation. Usually when I bring up compensation I’m met with a chorus of frustrated noises. But to me compensation is an opportunity to solve a complicated puzzle – and I really love a good puzzle. In this post I’m going to...
Edit 01/05/24: I just stumbled across a short YT video from Dr. Liston where AutoSpil is described in more specific details. I'm mad at myself. I remember seeing a bunch of tweets about something called AutoSpill back in 2021, and completely ignored it being the...
Should I use cells or beads? This question is at the center of many discussions nowadays in the flow cytometry world. Beads were once lauded as the easy alternative to cells to prepare your compensation controls. But as more and more researchers look at their...