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Facility News
This newsletter is the beginning of Volume 4 ! We've been sending you monthly bits of flow cytometry for 3 years already ! It is crazy to think about it and it is a pleasure to see you respond to it, ask for details and use it to develop your flow even further. Thanks again for the support ❤️.
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Our LSRIId got recently upgraded to a 5 laser machine ! There is now 3 UV channels (BUV395, BUV563 and BUV737) and we removed one channel from the Yellow Green Laser (PE-Cy5.5, happy farewell to you !). The full configuration can be found on the website >> LSRIId. As a consequence, note that your old experiments and templates won't work anymore and you will have to set up new experiments. If you need any help, please let us know.
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In this month FACS Tips, we are talking about autofluorescence and the benefits of spectral flow cytometry to deal with it efficiently. Enjoy the read 😊
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Sara Di Carlo won the mug this month, Congratulations !
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Each month, we will give away one of those special and unique mug designed by the FCF team. Answer correctly to our 3 questions and you can have a chance to win !
Please take few minutes to answer the quiz HERE.
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FACS Tips
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Autofluorescence
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Unfortunately, our fluorescent markers aren't the only thing to be excited under laser light. Rather, all living cells will fluoresce to some extent due to the presence of certain molecules, a phenomenon known as autofluorescence (AF). This fluorescence tends to appear strongest in UV and V channels but can exhibit unique profiles not only between cell types but also with different sample treatments, metabolic states, or cell growth stages. For these reasons, handling AF poses a complicated challenge. This is particularly true on full spectrum machines, which is also why Cytek, at the moment, has the most robust system to manage AF in our samples. Although not perfect, and certainly not simple, addressing AF can make the difference between obtaining valuable data and dealing with a smeared mess. Ignoring AF can lead to false positives and obscure dim resolving markers that fluoresce in similar channels..
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While certain samples are well known for having high AF, like skin and liver samples, spotting how AF may be affecting your samples can be tricky. One sign that it’s having an influence is the negative population is abnormally high on a dot plot. If you scan the NxN plots of your unmixed sample without any AF extraction and see negative populations sitting as high as 10^4 it could be sign that AF is playing a role. Another revealing pattern would be a strong diagonal signal in the UV and V channels or a multiple population presence in addition to the negative one. Autofluorescence also tends to spectrally align most similarly with fluorophores peaking around detectors UV7 and/or V7, so any markers peaking in these channels may have poor resolution. Markers with spectral signatures particularly similar to a typical AF spectra are BUV496, Zombie Aqua, Pac Orange, and FITC to name a few. Getting ahead of potential problems with AF in your sample may mean avoiding these colours if you know that AF is particularly high in your sample type. Once spotted correctly, this AF needs to be removed.
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Cytek has an evolving AF extraction process, and depending on how your sample looks, different approaches can be taken. While overly simple, try and categorize your sample under one of the three categories; Simple homogeneous, bright homogeneous, and complex heterogeneous.
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Simple homogenous autofluorescence: This is what you would tend to expect from a clean PBMC sample. It should be relatively dim and has just one spectrum. While it can certainly be advantageous to remove it, it shouldn’t have too strong an effect on your data if left in.
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Bright homogeneous autofluorescence: While still just one spectrum, this type is very bright, as high as 105 or higher in peak detector channels. This is certainly an AF that should be extracted, and is typical from neutrophils or macrophages. As it’s just one spectrum, using the built in AF extraction feature in Spectroflow as part of the unmixing is enough. It’s important when dealing with signals like this that you’re making sure that the AF you’re only extracting the AF for your cells of interest. For instance, if you’re working with whole blood for a T cell panel analysis, it’s important to only include your T cells and no other cell population in your AF extraction gates as it would otherwise mean multiple spectrums are potentially considered and it will interfere with the analysis of your goal cells.
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Complex heterogeneous autofluorescence: This is the most challenging type to manage, involving multiple unique signatures of varying intensities within a sample. A good example would be the analysis of myeloid population within a tumor. For situations like this, Cytek has developed it’s Autofluorescence Explorer tool. Even with the tool though it’s not a simple process. To find the various unique signatures, you need to examine the raw detector signals of your unstained sample, ideally with 50,000 or more cells recorded. From here look for 2 raw detectors with the greatest signal spreading potentially producing the greatest number of separate populations when plotted against one another. While UV7 and V7 can be great starting point it’s not always the best. For example, if you have Eosinophils in your samples one researcher suggested using YG1 detector to separate this autofluorescence along with V7. Gate as many unique populations as possible in the Autofluorescence Explorer, then they will be vetted for their similarity and any signals with less than 0.98 similarity can be used uniquely to unmix the sample. This process is quite complex and we expect we’ll expand on this in a future newsletter.
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This will certainly require some trial and error, and there is no one exact correct way to do this. Which certainly drives the subjective manor of this process. While subtracting one signature might work for an entire experiment, its also possible that different AF extractions will need to be done for each sample or patient within an experiment.
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This is certainly still a developing process and I anticipate as we expand our use of larger and more complex panels, Cytek, along with other spectral machine developers, will create better guidelines for how to properly remove AF in a more consistent way. This is certainly not an exhaustive description for all the tools and strategies for dealing with AF in your sample, and definitely feel free to reach out to the FCF staff or our Cytek technical support experts for better information n how to approach your individual samples. Additionally, I’ve included links to several webinars which were helpful in writing this newsletter.
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