Annexin-V - Cell membrane changes
In normal cells, phosphatidylserine (PS) residues are found in the inner membrane of the cytoplasmic membrane. During apoptosis, the PS residues are translocated in the membrane and are externalized. In general, this is not an early event in autophagy and the the role of PS externalization is unclear in autophagy. Interestingly the phospholipid, phosphatidyl- ethanolamine (PE) plays a crucial role in the formation of the autophagosome during the induction phase of the autophagic response in that it lipidates the microtubule LC3 in the double membrane of the autophagosome to form LC3B which is used as a marker for autophagosomes and hence the detection of autophagic cells.
The autophagic inducing agents, chloroquine and curcumin also induce apoptosis and thus PS is externalized to the outer leaflet of the plasma membrane and thus the annexin V assay can be used to detect these cells undergoing apoptosis during the autophagic response to chloroquine which ultimately blocks the autophagic response by it's chemical characteristic of being a weak base which inhibits the fusion of lysosomes to the autophagosome and thus inhibits the completion of the autophagic process.
The apoptotic process induced by chloroquine can be modulated by pre-treatment of cells with pan caspase blocker z-VAD, see figure. This causes a reduction in detectable annexin V binding cells and death of cells but an increase in the cells undergoing the autophagic process, see autophagosome section
Annexin-V is available conjugated to a number of different fluorochromes. Annexin-V is a specific PS-binding protein that can be used to detect some oncotic cells. Annexin-V is available conjugated to a number of different fluorochromes including Alexa Fluor-647 and FITC. Numerous DNA binding viability dyes can be used in the annexin V assay including PI, DAPI and DRAQ7(Biostatus).