Annexin-V - Cell membrane changes
In normal cells, phosphatidylserine (PS) residues are found in the inner membrane of the cytoplasmic membrane. During oncosis, the PS residues are translocated in the membrane and are externalized. In general, this is not an early event in oncosis and the the role of PS externalization is unclear in oncosis. In oncosis, annexin V is also externalized during the early phase of oncosis; the cells then rapidly proceed to cell death and losing membrane integrity, see figure. Oncosis is induced by heat shock (56C or 42C), sodium azide (1% solution), Triton X-100 or drugs used to induce apoptosis if used at higher concentrations can induce oncosis. Annexin-V is a specific PS-binding protein that can be used to detect apoptotic cells. 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, DAPIand DRAQ7 (Biostatus).
There are certain characteristics of oncotic cells that can be identified and used to detect oncotic cells in an otherwise healthy population of cells. The method employed to induce oncosis, either a) chemical; b) detergent; c) heat shock or d) drug generates a variable degree of DNA breakdown. The loss of DNA from permeabilised cells is due to DNA fragmentation. When cells are permeabilised, for example by 70% ethanol. 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 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 carbocyanine's, 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. In necrotic death, cells swell initially, and then cellular contents are rapidly released. This is seen on the flow cytometer as an initial increase in forward light scatter followed by a rapid decrease in both forward and 90o side scatter. Changes in cell size can be used in conjunction with surface phenotypic markers to identify the dying population .
Plasma membrane changes
In normal cells, phosphatidylserine (PS) and phosphatidyl-ethanolamine (PE) residues are found in the inner membrane of the cytoplasmic membrane. During apoptosis, the PS and PE residues are translocated in the membrane and are externalized resulting in a change in surface charge of the outer leaflet of the plasma membrane. In general, though not always, this is an early event in oncosis 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 by binding to PS.
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.
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. Until recently oncosis has been studied by the use of annexin V binding to externalized phospha- tidylserine (PS) residues which is exposed in relatively few cells undergoing cell death by oncosis in comparison to that observed in cells undergoing apoptosis.