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.
The fall in mmp can be measured flow cytometrically with a range of dyes all of which alter there fluorescence intensity with any change in mmp. These dyes include the carbocyanine molecules DiOC6(3) andDiIC1(5). Other cationic lipophilic dyes include CMXRos, TMRE and rhodamine 123.
Mitochondrial Permeability Transition Pore
The mitochondrial permeability transition pore (MPTP) is involved in the release of pro-apoptotic factors such as cytochrome c and Apoptosis Inducing Factor (AIF). The degree of permeability can be assessed flow cytometrically using dye Calcein AM.
Organelle Function - Reactive Oxygen Species
Reactive oxygen species include super-oxide, hydrogen peroxide and nitric oxide which are generated in small amounts during oxidative phosphorylation. Cells undergoing oxidative stress generate increasing amounts of superoxide and hydrogen peroxide.
There are numerous dyes available such as H2CFDA which are analogues of fluoroscein and dihydrogenated molecules of rhodamine 123 and Calcein AM that preferentially detect hydrogen peroxide.
Superoxide is preferentially detected by dihydroethidium and Oxyburst Green H2CFDA.
Nitric oxide is produced from L-Arginine by nitric oxide synthase. DAF-FM and DAF-FM acetate detect the presence of intracellular nitric oxide.