Quantitation of Apoptosis and Necrosis by Annexin V Binding, Propidium Iodide Uptake, and Flow Cytometry.
The surface of healthy cells is composed of lipids that are asymmetrically distributed on the inner and outer leaflet of the plasma membrane. One of these lipids, phosphatidylserine (PS), is normally restricted to the inner leaflet of the plasma membrane and is, therefore, only exposed to the cell cytoplasm. However, during apoptosis lipid asymmetry is lost and PS becomes exposed on the outer leaflet of the plasma membrane. Annexin V, a 36-kDa calcium-binding protein, binds to PS; therefore, fluorescently labeled Annexin V can be used to detect PS that is exposed on the outside of apoptotic cells. Annexin V can also stain necrotic cells because these cells have ruptured membranes that permit Annexin V to access the entire plasma membrane. However, apoptotic cells can be distinguished from necrotic cells by co-staining with propidium iodide (PI) because PI enters necrotic cells but is excluded from apoptotic cells. This protocol describes Annexin V binding and PI uptake followed by flow cytometry to detect and quantify apoptotic and necrotic cells.
Apoptosis and Cytotoxicity Laboratory, Mater Research, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia.
Lisa C
LC
Crowley
Apoptosis and Cytotoxicity Laboratory, Mater Research, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia.
Brooke J
BJ
Marfell
Apoptosis and Cytotoxicity Laboratory, Mater Research, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia.
Adrian P
AP
Scott
Apoptosis and Cytotoxicity Laboratory, Mater Research, Translational Research Institute, Woolloongabba, Brisbane, Queensland 4102, Australia.|Flow Cytometry and Imaging, QIMR Berghofer Medical Research Institute, Herston, Brisbane, Queensland 4006, Australia.|School of Medicine, University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia.
Nigel J
NJ
Waterhouse
D017304:Annexin A5 / Q000378:metabolism*; D017209:Apoptosis; D004396:Coloring Agents / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D009336:Necrosis; D011419:Propidium / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*
D017304:Annexin A5; D004396:Coloring Agents; D011419:Propidium