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

• Published in: Journal of immunological methods Vol. 423; pp. 99 - 103
• Main Authors: Pietkiewicz, Sabine, Schmidt, Jörn H., Lavrik, Inna N.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2015
• Subjects: Annexin A5 - metabolism; Annexin V/propidium iodide; Apoptosis; Apoptosis - physiology; biomedical research; Cell Death - physiology; Cell Line, Tumor; Death receptor; flow cytometry; Flow Cytometry - methods; Humans; image analysis; Image Cytometry - methods; Imaging flow cytometry; Jurkat Cells; Necroptosis; Necrosis - metabolism; Necrosis - pathology; phosphatidylserines; Phosphatidylserines - metabolism; propidium; Propidium - metabolism; signal transduction; Single-Cell Analysis - methods; staining; Staining and Labeling - methods; CD95L; MLKL; Imaging flow cytometry; Nec; DR; PCD; Necroptosis; Annexin V/propidium iodide; Death receptor; RIP; SFL; PI; Apoptosis
• ISSN: 0022-1759; 1872-7905; 1872-7905
• DOI: 10.1016/j.jim.2015.04.025

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. •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.