Single Staining and Compensation Controls in Flow Cytometry
Author: Mike Blundell | Reviewer: Chloe Fenton
As mentioned in Chapter 2 (Fluorescence Compensation) when performing multicolor flow cytometry, single stained samples are essential to determine the levels of compensation. Single staining will reveal the level of spectral overlap between different fluorophores and allow you to remove or compensate for this overlap (Chapter 2, Figure 12). This can be seen in Figure 21a, on Blocking Controls, where the fluorescence of FITC can be detected in the PE channel. Figure 21b shows how the data looks when properly compensated. This spectral overlap should be compensated for every fluorophore used.
Staining Rules
The important rules to remember when using single stained samples for compensation are:
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The staining of the compensation control must be as bright as or brighter than the sample. Antibody capture beads can be substituted for cells and one fluorophore conjugated antibody for another, as long as the fluorescence measured is brighter for the control. The exceptions to this are tandem dyes, which cannot be substituted.
Note: Although it would seem safe to assume that all tandem dyes created with the same donor and acceptor would have the same emission, this is not the case. Tandem dyes from different vendors or different batches must be treated like separate dyes, and a separate single-stained control should be used for each because the amount of spillover may be different for each of these dyes.
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The compensation algorithm needs to be performed with a positive population and a negative population. Whether each individual compensation control contains beads, the cells used in the experiment, or even different cells, the control itself must contain particles with the same level of autofluorescence. The entire set of compensation controls may include individual samples of either beads or cells, but the individual samples must have the same carrier particles for the fluorophores.
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The compensation control must use the same fluorophore as the sample. For example, both GFP and FITC emit mostly green photons, but have vastly different emission spectra. You thus cannot use one of them for the sample and the other for the compensation control.
- Enough events must be collected for the software to make a statistically significant determination of spillover. About 5,000 events for both the positive and negative population is ideal, but less can be used if necessary.
Frequently Asked Questions
What are compensation controls in flow cytometry?
Compensation controls are single-stained samples used to measure the level of fluorescence spillover from one fluorophore into other detection channels. They allow the calculation of compensation values so that spectral overlap can be corrected and true fluorescence signals can be accurately identified.
Why are single-stained controls required for compensation?
Single-stained controls are required because they reveal how much signal from one fluorophore is detected in other channels. This information is used to calculate compensation and remove spillover, ensuring that each fluorophore is measured accurately in multicolor experiments.
What makes a good compensation control?
A good compensation control must contain only one fluorophore, include both positive and negative populations, be as bright as or brighter than the experimental sample, use the same fluorophore as the test sample, and provide enough events for accurate analysis.
Can compensation controls use beads instead of cells?
Yes, antibody capture beads can be used instead of cells for compensation controls, provided they generate a suitable fluorescence signal. This is particularly useful when cell samples are limited or when it is difficult to obtain a clear positive population.
Why must compensation controls be as bright as or brighter than the sample?
Compensation controls must be as bright as or brighter than the sample to ensure that the full extent of spectral overlap is measured. Dim controls may underestimate spillover, leading to incorrect compensation and inaccurate data interpretation.
Why must compensation controls use the same fluorophore as the sample?
Compensation must be performed using the exact fluorophore used in the experiment because different fluorophores—even those with similar emission (for example, GFP and FITC)—can have different spectral properties and spillover characteristics.
How many events are needed for accurate compensation?
A sufficient number of events must be collected to allow the software to calculate compensation accurately. Around 5,000 events per population is typically ideal, although fewer events may be used if necessary.
What happens if compensation controls are set up incorrectly?
If compensation controls are incorrect, spectral overlap may not be properly removed. This can result in false-positive signals, inaccurate identification of cell populations, and unreliable experimental results.
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