Mitochondrial mASS AND FUNCTIONALITY

MITOCHONDRIAL MASS

Main compounds used to detemine mitochondria phenotype are:

• Nonyl Acridine Orange (NAO, ex 495, em 519)

• Mito ID Red (ex 558, em 690, fixable)

• Mitotraker Green FM (ex 489, em 517)

• Mitotracker Deep Red 633 (ex 644, em 665, fixable)

• Mitotracer Red (ex 581, em 644)

DETECTION OF CHANGES IN MITOCHONDRIAL MEMBANE POTENTIAL

Proper mitochondrial function requires a negative charge on the inner side of the mitochondrial inner membrane. A potential difference is maintained by the asymmetrical distribution of H+ ions across the membrane. Cationic lipophilic dyes such as JC-1, TMRE (tetramethylrhodamine ethyl ester), DiOC6(3), DiIC1(5), CMXRos (MitoTracker Red), LDS-751 and rhodamine 123 localize across the inner mitochondrial membrane. Loss of membrane potential is detected in cells by measuring a loss of dye from the inner mitochondrial matrix or a change in fluorescence of the dye as the transmembrane potential drops.

JC-1 is a cationic carbocyanine dye that accumulates in mitochondria. The dye exists as a monomer at low concentrations (e.g. in the cytoplasm) and emits green fluorescence, similar to fluorescein. At higher concentrations (obtained in mitochondria), the dye forms J-aggregates that exhibit an emission maximum at ~590 nm (orange). When mitochondrial membrane potential is lost JC-1 shows an emission change (orange to green).

The drop in TMRE accumulation in cells during the process of apoptosis is presented in the Apoptosis section.

Reactive oxygen species (ROS) detection

Reactive oxygen species (ROS) include a variety of molecules, starting from superoxide anion and hydrogen peroxide through singlet oxygen, peroxynitrite anion, hydroxyl radical and nitric oxide. These ROS molecules are produced both in normal and in disease conditions.

Superoxide can be detected by OxyBURST Green H2DCFDA or OxyBURST Green H2HFF BSA or mitochondrial superoxide indicator MitoSOX (ex 510, em 580). These dyes operate in a similar manner to the hydrogen peroxide sensitive dyes Carboxy-H2DCFDA, CMH2DCFDA and MitoPY-1 (mitochondrial Peroxy Yellow-1; ex 510, em 528/540). Their fluorescence increases when particular reactive oxigens species are present.

Similarly, dihydrorhodmine 123 is oxidized preferentially by hydrogen peroxide, then the free rhodamine 123 signal can be detected as it fluoresces upon binding to mitochondrial membranes.

Nitric oxide is produced during the process of apoptosis from L-arginine by the action of nitric oxide synthase. This form of ROS may be detected by loading cells with fluoroscein analog DAF-FM, which fluorescence increases upon binding to nitric oxide.