NucView®488 and RedDot™ Apoptosis/Necrosis Assay Kit contains NucView®488 Caspase-3 Substrate for detection of caspase-3/7 activity. The far-red dead-cell stain RedDot™ is included for staining necrotic and late apoptotic cells that have compromised plasma membrane integrity. This kit provides a convenient tool for profiling apoptotic and necrotic cell populations by fluorescence microscopy or flow cytometry.

In contrast to other fluorogenic caspase substrates or fluorescent caspase inhibitor based (FLICA) assays, NucView®488 Caspase-3 Substrate can be used to detect caspase-3/7 activity within individual intact cells without inhibiting apoptosis progression. The substrate consists of a fluorogenic DNA dye coupled to the caspase-3/7 DEVD recognition sequence. The substrate, which is initially non-fluorescent, penetrates the plasma membrane and enters the cytoplasm. In apoptotic cells, caspase-3/7 cleaves the substrate, releasing the high-affinity DNA dye, which migrates to the cell nucleus and stains DNA with bright green fluorescence. Thus, NucView®488 Caspase-3 Substrate allows detection caspase-3/7 activity and visualization of morphological changes in the nucleus during apoptosis.

RedDot™ is a cell membrane-impermeable, far-red dye with high selectivity for membrane compromised or dead cells. RedDot™ has far-red emission at 695 nm for detection in the Cy®5 channel, well-separated from the green fluorescence of NucView®488. The excitation maximum of RedDot™ is 665 nm, but it can be efficiently excited by wavelengths from 488 to 647 nm, and therefore can be used with the 488 nm flow cytometry laser line.

Note: while NucView®488 staining is formaldehyde-fixable and compatible with subsequent immunostaining, fixation after staining with RedDot™ is not recommended because it can result in increased background staining of healthy cells.

NucView® enzyme substrate technology is covered by U.S. patents.