Hi everyone,
I want to have an advice for the analysis of flow cytometric analysis of cells by the use of fluorescence intensity and front scatter. I stained yeast cells and want to analyze the size dependent fluorescence signal. Is it correct to divide this signal (525-A or 585-A) by the front scatter signal (FS-A)?
Best
Marco
Hello,
I don't know how the yeasts look like in flow, but maybe you should divide the cells into different subsets according to their FSC (and maybe SSC also) and then compare the MFI of those subsets? You may also think about the staining index.
FS is usually only used as a gating strategy, rather than quantifiable data.
I assume you do not have separate size populations of cells, and it is more of a scatter. It may be a viable choice to arbitrarily divide your yeast cell population into quartiles. You could then take the median florescent intensity for each size quartile.
Unless you have distinctly different size populations it is difficult to attribute a single cell size with its corresponding fluorescence. Either way, you dont need to divide by FS.
To echo what Lewis said; FS and SSC are rarely used for quantification, mostly for gating. If you do want to use FS for quantification, a violet or UV laser should give you better resolution, thus allowing better discrimination of any yeast size subpopulations. Its also important, if quantifying on size properties, to make sure threshold setting is well optimized using both yeast samples and cell negative control (everything but the yeast cells).
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