Autofluorescence Controls...

Are most appropriate when your samples may exhibit naturally high levels of autofluorescence that may differentially impact the sensitivity of resolution in each detection channel.

Different cell types and tissues have varying levels of inherent fluorescence. Major sources of autofluorescence include NADH, riboflavin, metabolic cofactors, the crosslinking of primary amines by paraformaldehyde, and certain biological structures (e.g., mitochondria, lysosomes). These proteins and molecules are more easily excited at lower wavelengths (i.e. from the UV, violet, and blue lasers) and will emit at a wide range of 300-600 nm, which overlaps with several common fluors like BV421™, Pacific Blue, and FITC. Myeloid cell lineages tend to be particularly autofluorescent. Furthermore, stimulating cells can cause them to become more metabolically active, producing more autofluorescent proteins, vitamins, and cofactors. An unstained control sample is helpful in delineating how much autofluorescence populates your channel of interest. Also, autofluorescence doesn’t have a large Stokes shift (the difference in nanometers between the peak excitation and emission wavelengths), so tandem fluorophores can prove very useful to resolve populations on highly autofluorescent cell samples. If necessary, these factors can be taken into consideration when building a flow panel.