I am performing a project using flow cytometry and I cannot find the proper gating technique of the FL2 settings. If anyone has any advice or help, that would be greatly appreciated.
Article Genome Size Determination Using Flow Cytometry of Propidium ...
i dont know about plant cells, but they might be fluorescent enough on their own to interfere with a gating purely in FL2.
first you gate FSC and SSC to have only single cells, then you compare the stained (PI) and nonstained (control) cells (per experiment you need at least one control). once you have their histograms, just gate that none of the non-stained are counted and apply the gate to all samples then.
Hi Lauren,
The whole game depends on the software and the machine. I estimated genome sizes using BD FACSVerse.
If you just want to assess the genome size, I think you do not need to gate your nuclei based on FS or SC, rather I would suggest you to focus more on the dot plot PI-A vs PI-W in log scale to set the voltage properly and to do the doublet discrimination.
As plant cells create a lot of debris, it is very important to prepare good quality of samples, and identification of proper nuclei populations are crucial.
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