Measuring intensity of overlapping proteins



I am very new in using cellprofiler and I got some problem with analyzing some staining. I stained my samples for CD31 and a second protein. I would like to quantify the intensity of my second protein only in the areas, that are overlapping with my CD31 postitive cells. By doing that I would like to see the expression of my second protein in endothelial cells (CD31+ cells).

So far I was only able to get good quantifications of my separated staining with the IdentifyPrimaryObject tool. I also figured out, that I can relate my objects. I chose CD31 as parent and the second protein as child. I continued using the FilterObjects tool to measure the Slit2, that are overlapping with the CD31. My problem is, that the quantification really doesn’t give the results, that I am aiming for. Cellprofiler seems to count the cells, that are overlapping. But what I want is: to substrate all the areas of my second protein, that don’t overlap with CD31 and quantify the expression of the protein in the overlapping areas.

Did somebody tried to do the same before or has ideas how to solve that problem?





It seems that you can Identify CD31+ objects (let’s name them “CD31_cells”) From that step, you can simply add module “MeasureObjectIntensity” , in this module :

Select an Image to measure choose “Slit2” (named after the protein channel)
Select object to measure choose “CD31_cells”

This will measure only the region enclosed (overlapped) by CD31 signal, as you wanted.

Unless there is a misalignment of signal between CD31 and Slit2, you don’t really need to relate objects.



Hi Minh,

thanks a lot for your respond. I am curious, whether this function will really measure the Slit2 expression in the overlap, because my staining in two different images. I think it is best, if I show you briefly, how I set up the program till now.

Are the data, that I receive really the overlap? what do you think?




Hi Simon,

Thanks for clear screenshots.

In your pipeline, it’s not necessary to have 2 x IdentifyPrimaryObjects, the later one (identify CD31_pos) is sufficient.

But ! In your MeasureObjectIntensity :

Select an image to measure : please choose "Slit2"
Select objects to measure: please choose “CD31_pos”

That will assure : everything you measure will be Slit2 intensity signal, within the boundary of CD31_pos objects.


Awesome! Thank you so much!!