Cellular membrane asymmetry loss is seen as an early sign of apoptosis, where phosphatidylserine (PS) residues embedded in the inner plasma membrane become externalized and signal phagocytosis.
A human placental protein called Annexin V is found to bind PS specifically when calcium is present. One tool used very commonly is fluorochrome-conjugated annexin V, which is capable of the detection and quantification of this type of loss of membrane asymmetry, with the characteristic exposure of PS.
Many experiments use both annexin V binding and other reagents for cell viability like propidium iodide (PI) which do not usually show the ability to pass through the cell membrane, to distinguish apoptotic cells from necrotic ones.
Detection of Fluorochrome-Conjugated Annexin V
When fluorochrome-conjugated annexin V binds to exposed PS residues, it can be detected using either fluorescence microscopy or flow cytometry. The first technique allows clear visualization of the binding, but the second is more useful as it can be used to quantify cells quickly and accurately, using the exposed PS residues.
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.
Reversible Annexin Probe
If the cell is undergoing physiological stress or is inflamed, PS residues are exposed, again, but cell death is not inevitable. The issue here is that annexin V binds to these sites irreversibly, and this could lead to an excessive estimation of the number of apoptotic cells. To prevent this, Abcam has a reversible annexin probe that is capable of detaching from PS once the molecule turns back to the original location in the inner layer of the plasma membrane.
This enables apoptosis to be visualized in living cells over the course of time. Further discrimination of necrotic and apoptotic phenomena can be accomplished using propidium iodide (PI) or other similar nuclear dyes, which cannot enter viable or apoptotic cells, as the figure shows.
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