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.
DiIC is imaged in sorted dead cells counterstained with DAPI andsorted live cells
Imaging 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, though not always, this is an intermediate 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. Annexin-V is available conjugated to a number of different fluorochromes. Theannexin-V positive and dead cells can be sorted and imaged by confocal microscopy.
- Sorted dead cell showing reduced mitochondrial function
- Sorted live cell showing active mitochondria
- Sorted apoptotic cell stained with annexin V-FITC
- Sorted dead cell stained with annexin V-FITC and DAPI
Imaging DRAQ7 with Annexin V
The new Biostatus viability dye DRAQ7 is excited at 488 and 633nm and emits at >665nm can be combined with annexin V-FITC to detect apoptotic cells, see figure.