Dealing with Autofluorescence
What is AF?
Strategies for dealing with it
Resources
Multi-AF
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Dealing with autofluorescence (AF) effectively requires knowing what it looks like and designing your panel around it. Traditional flow cytometry approaches often incorporate "dump" channels or live/dead discrimination into areas of high AF, which is a decent strategy if you don't intend to extract the AF. For spectral flow, we often prefer to unmix the AF profile(s) as separate channel(s) in the data, removing that spectrum from our data to improve resolution. However, if the AF profile is not considered when designing the panel, this may lead to an unintended loss of resolution due to unmixing-dependent spreading error.
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To combat this, we must design our flow panels with the intrinsic AF profiles in mind. If you plan to unmix AF, be sure to leave space in spectrum to do so. To that end, here are some AF profiles of common immune cell types, as seen by a 5-laser Aurora. These profiles will be fairly stable under homeostatic conditions. They will change with age, inflammation, disease or activation status.
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Human PBMCs
With human PBMCs, the AF profiles are pretty much the same in lymphocytes and monocytes. There's no point trying to separate these out. The intensity is stronger in monocytes, but not enough to reliably gate on.
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Similar fluorophores: BV510. To a lesser extent, BUV496 (UV7), Live/Dead Blue (fixable UV, Zombie UV), StarBright UV 445 for UV6 and Real Blue 545, Spark Blue 550 or AF532 for the B3 channel. Using all of StarBright UV445, BUV496, BV480, BV510 and StarBright Violet515 with automated AF extraction will cause unmixing-dependent spreading error.
Mouse Splenic Macrophages
Where do we see fluorescence? The peak lies in V10, the same place as BV605. There's additional signal in YG7 (PE/Fire 700 or PE-Cy5.5), widely across the blue laser, and in V8 (think BV510, Pacific Orange). This signal is bright enough that you can use it to separate out and identify the cells, if you really want to.
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If you want to explore expression of markers on mouse macrophages, avoid putting low expression targets in those channels. If you want a clear area in order to identify and remove AF contamination from splenic macrophages, leave a channel unoccupied in the area of V7-V15.
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Mouse Microglia
Remarkably similar to splenic macrophages. Stronger in intensity. The AF of microglia is definitely enough to gate on.
Mouse Alveolar Macrophages
Very different. A strong UV signature. You'll want to be careful using BUV496 (UV7), StarBright UV445 (UV6), and BUV563 (UV9).