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 externalised. In general, though not always, this is an early event in apoptosis and is thought to be a signal to neighbouring cells that a cell is ready to be phagocytosed. Annexin-V is a specific PS-binding protein that can be used to detect apoptotic cells.
There are a number of caspases in mammalian cells that have been shown to be involved in the early stages of apoptosis, e.g. Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9 and Caspase 10. The functions of these enzymes are not yet entirely clear, but it appears that after an initial signal to the cell to undergo apoptosis, they may be responsible for the activation, amplification and execution of the apoptotic cascade. Because of the central importance of the caspases in apoptosis, their detection by flow cytometry has become widespread. The activity of enzymes implicated in apoptosis may be detected in three ways:
There are certain characteristics of apoptotic cells that can be identified and used to detect apoptotic cells in an otherwise healthy population of cells. Technically the easiest characteristic to detect is loss of DNA from permeabilised cells due to DNA fragmentation. When cells are permeabilised, for example by 70% ethanol, the fragmented 182bp DNA multimers leak out of the cell. The result is a population of cells with a reduced DNA content. If the cells are then stained with a DNA intercalating dye like propidium iodide, then a DNA profile representing cells in G1, S-phase and G2M will be observed with apoptotic cells being represented by a sub G0/G1 population seen to the left of the G0/G1 peak.
Imaging mitochondrial membrane potential changes
Commonly employed reagents and cell treatments, such as staurosporine, etoposide and UV-B irradiation cause mitochondrial membrane potential (mmp) to reduce before phosphotidylserine (PS) flipping. These changes in mmp can be detected by a range of fluorescent dyes, including carbocyanines, DiO, DilC, and TMRM and MitoTracker Red (CMXRos). All these dyes show a fall in fluorescence as mitochondrial membrane potential falls during apoptosis.
A dot plot of forward light scatter versus 90o side scatter is a measure of cell size and cell granularity respectively, the latter being dependent upon the presence of intracellular structures that change the refractive index of light. Cell condensation during early apoptosis is seen as a decrease in forward light scatter only. As apoptosis proceeds, both
Organelle Function - Mitochondrial Membrane Potential (mmp)
The mmp is due a differential distribution of proteins on either side of the impermeable inner mitochondrial membrane. In apoptosis,irrespective of the stimulus, loss of mmp occurs with the formation of mitochondrial permeability transition pore (PT) or mitochondrial megachannel.