The detection of, and discrimination between, apoptosis and necrosis can be accomplished by differential staining of markers characteristic of the relevant type of cell death, for example, with Hoechst 33342, propidium iodide and Annexin V-FITC.
Hoechst 33342 (blue) – nuclei stain
Propidium iodide (red) – necrotic cell stain
Annexin V-FITC / Alexa Fluor® 488 (green) – binds to the early apoptosis marker phosphatidylserine
Using a proprietary algorithm, the software can uniquely identify three object classes:
- Blue objects – cell nuclei
- Blue/red objects – necrotic cells (for live:dead cell ratio)
- Blue/red/green objects – apoptotic cells (for apoptotic:necrotic cell ratio)