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Facility News
Hello all ! I hope this newsletter will find you on your return from vacation in a couple of weeks. As much as we love the flow, it is nice to take a break once in a while :)
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This month, we talk about the Fluorescence Minus One or FMO control ! They are the best controls in town for your precious and hard to define population of interest. They will multiply your tubes and control wells in the first few experiments but in the long run will help produce those sweet data your PI is yearning for ! So please, remember to be smart about it and take it slowly !
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In the Flow Post-it ! We explain the impact of autofluorescence on your data. A problematic inherent fluorescence that can be removed with a few tricks. May we remind you as well that the Cytek Aurora has this lit feature that can isolate the autofluorescence ? If only someone could train you to use it in the facility, uh ?
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We will be here in August so stop by and enjoy a relaxing training on the cool Aurora flow cytometer now that you followed the theory last month ! It's not like the sun is going to bother us !
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FACS Tips
Fluorescence Minus One (FMOs)
Placing gates correctly can be one of the hardest things in flow cytometry, and it’s made harder when we do not use the appropriate controls. While certain fluorophores have distinct On/Off expression patterns, creating clear separation, others appear as a smear moving from negative to positive without clear distinction. Using the correct controls can overcome these problems and improve your data.
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Unstained Control
Relying on just an unstained control may work for some fluorophores, but for larger panels they become increasingly unreliable, and unless they match the activation state and autofluorescence of the cells being examined they can be ineffective for gating.
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Adapted from Bushnell, T., & Kissner, M. (2018). Advanced Topics in 4-10 Color Compensation for Flow Cytometry [electronic resource] (1st ed. 2018.)
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Isotype Control
If we want to account for any background non-specific staining, isotype controls become a valuable tool. Isotype controls, acting as a manufacturing control for the antibodies, are specific for a protein not found in the target species* and therefore should produce no signal. General non-specific binding problems can be determined with this approach; however, it doesn't account for the spread associated with spectral overlap between fluorophores that can push your negative population closer to the positive in stained samples. Isotype controls should also be titrated properly too, adding another layer of complexity if they are to be used properly.
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*Fun Fact: Often the KLH antigen is used as this control protein. KLH is a protein antigen found in the giant keyhole limpet, an aquatic organism, not found in mice or humans.
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FMO Controls
Take all the fluorophores you need to stain with, then subtract one, and you have your FMO. These controls provide the best method for gating positive cells. They account for the spreading due to spillover of fluorophores that exist even in well compensated samples. This reduces the possibility of including false positive events in your data analysis. Having an FMO is not necessary for every channel, or even every experiment, but is certainly a useful step when first optimizing an experiment to have an idea to the extent of spillover/spreading of fluorophores in your panel. It is still necessary to use an Fc block when staining samples, even when you have FMO controls, as it's important to reduce any non-specific staining when possible.
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Conclusion
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FMO controls are a great way to take out some of the error and bias in gating, especially on some of the more complicated panels.
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Flow Post-its
Social media is a great source of information even when it comes to flow cytometry. One useful resource is the twitter account of the Flow Cytometry Core Facility at Memorial Sloan Kettering Cancer Center (https://twitter.com/FlowMskcc). We thought it would be nice to highlight their tips and bring them to you !
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