Detection of loss of membrane asymmetry in apoptosis

The loss of cellular membrane asymmetry is considered an early sign of apoptosis, wherein phosphatidylserine (PS) residues embedded in the inner plasma membrane become externalized and signal phagocytosis.

Annexin V, a human placental protein that specifically binds to PS in the presence of calcium, and fluorochrome-conjugated annexin V in particular, is a commonly used tool to detect and quantify the PS exposure characteristic loss of membrane asymmetry.  The binding of fluorochrome-conjugated annexin V to exposed PS can be detected by flow cytometry or fluorescence microscopy. While fluorescence microscopy will allow the visualization of the event, flow cytometry is the most useful method as it allows for a quick and accurate quantification of cells with exposed PS.

ab14085 Annexin V-FITC Apoptosis detection kit
Annexin V binds to exposed PS irreversibly. Cellular conditions such as physiological stress or inflammation can lead to PS exposure but not necessarily to cell death. To address this problem, we also offer a reversible Annexin probe that can detach from PS when the molecule flips back to the inner membrane.
This reversible Annexin probe will allow you to investigate apoptosis in live cells over time. To further distinguish apoptosis from necrosis, cells can be stained with nuclear dyes such as propidium iodide (PI) that are excluded from entering viable and apoptotic cells.

Annexin V-FITC Apoptosis Detection Kit (ab14085): AG06173 primary fibroblasts were incubated with Annexin V-FITC for 15 min in the dark. Propidium iodide was used as a counterstain to discriminate necrotic/dead cells from apoptotic cells.  Left image:  positive control AG0613 cells irradiated at 10Gy; right: negative control AG06173 untreated cells.

​​​Annexin V detection should be paired with the use of cell viability reagents such as propidium iodide (PI) or 7-AAD, which are not able to penetrate the plasma membrane and can be used to differentiate between  apoptotic and necrotic cells. The table below shows a quick representation of how to interpret the data obtained from annexin V and viability staining.

Low Annexin VHigh Annexin V
Low viability dye
viable cellsapoptotic cells
high viability dye
dead cellslate apoptotic/ necrotic cells

Phosphatidylserine exposure can also occur in other types of cell death such as necroptosis. Therefore, loss of membrane asymmetry should be considered more a confirmatory assay for apoptosis than a defining assay and should be run alongisde other apoptosis assays.

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