I am doing Hoechst stain to detect apoptosis in my cells. Apoptotic cells have condensed chromatin which would be stained brighter than that of normal healthy cells. However, when cells undergo mitosis, their chromatin also condensed, So how do we differentiate the condensation and more brightly stained chromatin between apoptotic or mitotic cells?
Flow cytometry assays also show that about half of the cell population are in G2/M arrest. so, in this case, is Hoechst staining a good way to detect apoptosis?
Lee
You’re asking a very insightful and important question, and you’re absolutely right to question the specificity of Hoechst staining in distinguishing apoptotic cells from mitotic cells — both show chromatin condensation and brighter Hoechst fluorescence.
Hoechst staining alone is not a good way to detect apoptosis in G2/M arrested cells?
— because mitotic condensation mimics apoptotic chromatin in fluorescence intensity.
You should always combine Hoechst with morphology assessment and/or apoptosis-specific markers.
Under the microscope look for following features:
Apoptotic nuclei: Condensed fragmented, irregular, often crescent- or ring-shaped.