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As mentioned in Chapter 2 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 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.
The important rules to remember when using single stained samples for compensation are: