Quantification of apoptosis and necroptosis at the single cell level by a combination of Imaging Flow Cytometry with classical Annexin V/propidium iodide staining

• Apoptosis and necroptosis are two major programs of cell death.
• Method of the ultimate quantitative distinction between programmed necrosis and apoptosis.
• Method uses classical Annexin V/propidium iodide staining.
• Method is based on the Image-based analysis of nuclear morphology of the cell death.

Precisely identifying the type of programmed cell death is one of the key questions in contemporary biomedical research. We developed a straightforward approach allowing quantitative discrimination between two types of cell death on the single cell level: apoptosis and necroptosis. This method uses the combination of imaging flow cytometry with classical Annexin V/propidium iodide staining, which allows for the ascertainment of typical features of dying cells: exposure of the phospholipid phosphatidylserine and the loss of membrane integrity. Image-based analysis of nuclear morphology enables us to distinguish between secondary necrotic/late apoptotic and necroptotic cells directly in one assay. This is a major advantage compared to other contemporary approaches of necroptosis detection, which require a parallel application of several methods. This approach can be used for the quantitative assessment of cell death in cell and systems biology studies of signal transduction networks.