Propcytoplasm Vs distcytoplasm

cellprofiler

#1

Dear Team,

Since,nuclear translocation assay gives you distance cytoplasm as well as propagated cytoplasm, I plotted two scatter plots of green florescence in nuclei (y-axis) vs either type of cytoplasm (x-axis).

I have attached the two plots.
There are remarkable differences b/w the two for same measurements except the type of cytoplasm used.
I have two queries in this regard:

  1. What conclusion can be made from these plots?
  2. Which type: should we use for further study?

Plz help so that correct conclusions can be drawn out.
Thanks
Mridul KK


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#2

There are two ways to address your question:

  1. Qualitative: You can look at the Propagate-Cytoplasm outlines on your images and the Distance-Cytoplasm outlines on your images and decide: which ones consistently define the region where you expect to see cytoplasmic green fluorescence? Ideally, you are running the Propagate option in IdentifySecondary on a cytoplasmic marker stain image that is distinct from your green translocation marker image. If that is not the case, your strategy has somewhat circular logic: you are defining the cytoplasm based on a marker which in some samples will be entirely nuclear! If you do not have a separate marker for the cytoplasm/cell body (like actin or tubulin for example), you must choose the Distance option.
  2. Quantitative: If both options yield outlines that seem to reasonably mark the cytoplasmic area where the green marker might be located, then you can quantitatively compare the results of the two options. Gather several positive and negative control images (ideally at least 10 of each) where the green is cytoplasmic or nuclear, respectively. Then add the CalculateStatistics module to the end of your pipeline, after measuring the nuc/cytoplasm ratios using both Propagate and Distance options. CalculateStatistics will tell you which option yields the most clear distinction between your positive and negative control samples. For help on using CalculateStatistics, see the recent tutorial called “Step-by-step CellProfiler tutorial, using human cell cytoplasm-to-nucleus translocation as an example”: cellprofiler.org/examples.htm

Good luck,
Anne


#3

Dear Anne,
Just saw your reply after a long break (due to hurricane IKE disaster).
Thanks for your nice explanation. I’ll definitely look into it.
Mridul KK