The Do's and Don'ts of Compensation

Posted June 4, 2019

Compensation is the process of correcting for spillover, when one fluorophore is detected in multiple channels.  It is required for most experiments of four or more colors to identify the correct signal that should be measured in each channel.  This quick list can help new flow cytometry researchers navigate the basics of compensation.

 

1. DON’T – Panic!  Compensation may seem intimidating at first, but modern flow cytometry software can make automated compensation a relatively straightforward process.  Also, be sure to ask your flow core manager for their recommendations if you have any questions.

 

2. DO – Run compensation controls.  Single-color compensation controls containing both positive and negative populations should be tested for each fluorochrome.

 

3. DO – Use the exact same fluorophores for your compensation controls that you will use to stain your experimental samples.

 

4. DO – Prepare your control samples in the same way (ie. fixation, permeabilization treatment, etc.) as your experimental samples.

 

5. DO – Use the same cell type for both positive and negative control populations to prevent variations in background auto-fluorescence levels.

 

6. DO – Choose bright controls. The positive population should comprise at least 10% of the total cell population and the signal must be at least as bright as any of the fluorophores that will be used in the experiment.  Brighter is generally better, so long as the signal is within the scale of detection.

 

7. DO – Run freshly stained compensation controls for each experiment, even if you have previously generated similar compensation matrices. Fluorescent dyes can lose potency over time or may even vary from lot to lot.

 

8. DON’T – Count dead cells. Use viability dyes to exclude any dead cells and debris from your analysis, as they can skew data and lead to improper compensation.

 

9. DO – Reduce spectral overlap during your panel design. Use FluoroFinder’s Panel Design Tool to make dye selections that spread your fluorescent signals.  Then compare your dye selections on the Spectraviewer and see how they will be detected in each channel on your cytometer.

 

10. DO – Balance your PMT voltages. Use stained cells to optimize, as your cytometer’s default settings may not be optimal for your sample.

 

Follow these ten tips and compensate your next flow cytometry experiment with confidence!

 

 

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