I am working on phosphoflow in FACS and have stained the cells with surface markers and intercellular markers. I got the FSC and SCC results and histogram but I am confused about how to interpret the data. What should nonphosphorylated and phosphorylated proteins looks like in in the histogram?
Hi Komal,
I find your question a little strange, but I'll try to answer it :)
In my text I'll just use Smad3 and phospho-Smad3 as examples.
Regarding how the histograms should look, you can't really interpretate one histogram: you need to compare different histograms. This can either be done within the same experiment (e.g. the Smad3 histogram for CD4 cells vs. the Smad3 histogram for CD8 cells) or as different experiments (e.g. the phospho-Smad3 histogram of stimulated cells vs. unstimulated cells).
When you just look at the histograms, they may actually look fairly similar since intracellular proteins often are present in all cells. For the same reason, you would usually report the mean fluororescence intensity in different populations. A very common plot is the histogram overlay - I have made an example with some Treg markers that you can look at. It is the same experiment (same test tube), but the populations listed on the left are defined according to "standard" gating.
The plot was compiled using cellbank.org (it is free).
Thank u thomas. comparission with diffrent histogram of the same experiment is good to understand how phosphorylation looks in the histogram
Dear Komal,
Staining for flow cytometry would be seen in fluorescent channels not in FSC to SSC.
Comparing FSC to SSC channels would tell you if cell undergo physical changes (which could be part of your experiment) but have nothing to do with staining. Since, as you say you did intracellular staining, FSC to SSC features of your cells will be changed (in comparison to living fresh cells) due to your procedure as most intracellular staining employ some kind of porosive fixative agent e.g. formaldehyde etc.
Another advice, if you can, try to analyze your results using 2d plot such as dot plot density plot or contour. It is much more efficient for multiple staining.
Good luck
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