Count double and triple labeled cells

I downloaded CellProfiler yesterday and I want to make certain that I am doing things correctly. I want to quantify the number of triply stained neurons I have grown in culture. They are stained with DAPI, NeuN as a cell-type marker, and cleaved Caspase3. As one of my stains is a little dirty, I want to make sure that any NeuN or Capase objects map to a DAPI object. My goal is to come the grid below.

          | caspase - | caspase + |
   NeuN - |     A     |     B     |  A+B=E
   NeuN + |     C     |     D     |  C+D=F
          |   A+C=G   |   B+D=H   | E+F=G+H=I

I = all DAPI positive cells 
F = NeuN+DAPI+ cells
H = Capase+DAPI+ cells
D = NeuN+Caspase+DAPI+ cells

The above is what I am trying to measure, and everything else can be easily calculated. I think I am doing this correctly, but want to make certain I am not missing something. Any suggestions?

Thanks in advance.
04082015C-pipeline.cppipe (34.5 KB)

I think you have much the right idea. I’m attached a revised pipeline, in which I did the following:

  • I replaced RelateObjects with MaskObjects, as RelateObjects enforces that an overlapping child object can only be assigned to one parent, whereas MaskObjects can assign the same overlapping object to multiple parents.
  • The FilterObjects strategy is otherwise the same as yours.

As long as you are assured that the NeuuN and Capase identification is sufficient, the basic approach should work fine.

assay.cppipe (23.4 KB)