My project consists in quantifying the levels of a transcription factor in the nucleus and cytoplasm. Because the transcription factor is also in the cytoplasm, I have been able to use its staining to define the cells and the outer limits of the cytoplasm (identify secondary objects) after having identified the nuclei (via identify primary objects). I then identified tertiary objects (cytoplasm) and got the values for the transcription factor in cytoplasm vs. nuclei.
My experimental setup will change in a way that I will have, in the same picture, 2 different cell types that I’d like to analyze as described above, with the added data that the 2 different cell types have a different surface staining (cell type A is stained with BV421 and cell type B is stained with Pe-Texas Red). How do you suggest that I build the pipeline so that it distinguishes between the two cell types (via a mask?), defines the outer edge of the cell, and quantifies the transcription factor expression in cytoplasm vs. nucleus. (Cytoplasm can still be defined as done previously using the transcription factor staining).
Thanks in advance for your help in this!