I am a complete beginner in flow cytometry and I work with yeast (S. cerevisiae) expressing GFP from a chromosomally integrated construct. I want to compare the coefficient of variation of GFP expression in different strains. I understood that the gating is a very important step and I want to be sure to do it properly. From what I understood, in theory, I can distinguish in between singlets and doublets plotting SSC-H vs SSC-W then FSC-H vs FSC-W. But what I obtain is not as clear as what I could see in videos/webpages explaining how to do so. Also, I don't know if it is easy to distinguish in between doublets and budding cells with this method or if it requires an additional DNA stain. In the lab, we try to sonicate our cells to get read of aggregates/doublets, but our strain is particularly prone to aggregation so it is not always super efficient. I attached an example for one strain where I attempted to identify diverse populations. Could somebody with experience in yeast flow cytometry help me interpret it? For example, does my P1 population corresponds to non-budding and budding cells or singlet and doublets and singlets is just P2? Is the shoulder visible on the right side of TEF1-L0 sample composed of aggregates but if I can remove it with a gate, then I end up with the singlet population to analyse? Similarly, if I try to gate according to SSC-H vs SSC-W, it does look like there are two main populations, but again, is it singlet vs doublet or non-budding vs budding cells?
Many thanks in advance for your help.
Estelle,
I'm by no means an expert in yeast flow cytometry, but in my limited experience, trying to distinguish between budded cells and doublets is pretty much impossible by forward- and side-scatter gating. This is because there is a lot of cell size variability amongst individual cells (even when genetically homogeneous), and the two groups overlap a lot in the SSC and FSC distributions. I think your best bet is to also stain the DNA and gate by genome copy number. If you are fixing cells, this should not be a problem as there is virtually no overlap between propidium iodide and GFP spectra (SYTOX green is best for DNA, but won't be possible in your case). If you are analyzing live cells, this might be a problem - I don't know of any vital DNA stains that work efficiently with yeast (but I haven't researched this very much).
The alternative is to disrupt the cell clumping with mild treatments such as brief zymolyase or detergent in a sorbitol solution. Is your strain flocculating or does it have a septation defect? The best treatment will depend on the cause of cell clumping. Again, I can't remember the details, but I know there are ways to reduce flocculation with ionic or mild detergent disruption. A septation defect will require enzymatic treatment. Hope someone more knowledgable responds and good luck.
Estelle, I am following your question because I find this subject very interesting. I don't have any experience with yeast flow cytometry but I found this article that maybe can help you. Apparently if you sonicate your sample for 5min using an ultrasonic water bath you could minimize the amount of cell doublets. Take a look at the paper. I hope it can help you. If you find a good method please update us. GOOD LUCK!!
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017175#s3
Hello Estelle,
By no means i am an expert in yeast flow cytometry, but i was thinking that doing image flow cytometry might help. As in those systems one can have visual of the proccessed cells maybe the discrimination of douplets from budding yeast cells would be easier, in combinattion of course with their scatter properties.
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