NucView™ 530 Caspase-3 Substrate, 1 mM in DMSO, provides a convenient tool for detecting apoptosis in intact cells based on caspase-3/7 activity using either confocal microscopy or flow cytometry.
In contrast to other fluorogenic caspase substrates or fluorescent caspase inhibitor based (FLICA) assays, NucView™ caspase-3 substrates can be used to detect caspase-3/7 activity within individual intact cells without inhibiting apoptosis progression. NucView™ substrates consist 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 fluorescence. Thus, NucView™ Caspase-3 Substrates are bifunctional, allowing detection of caspase-3/7 activity and visualization of morphological changes in the nucleus during apoptosis. The staining is also formaldehyde-fixable.
NucView™ 530 Caspase-3 Substrate stains apoptotic cell nuclei with orange fluorescence (Ex/Em 528/563 nm, Figure 1), for detection in the Cy®3 channel by fluorescence microscopy (Figure 2) or the PE channel by flow cytometry (Figure 3). NucView™ 530 can be used for multi-color imaging with blue, green, or far-red fluorescent probes. Note that when excited by the 488 nm laser line, NucView™ 530 also fluoresces in the FITC channel, and therefore cannot be analyzed together with green probes by flow cytometry.
Biotium also offers blue fluorogenic NucView™ 405 Caspase-3 Substrate, and green fluorogenic NucView™ 488 Caspase-3 Substrate and kits.
Figure 1. Excitation and emission spectra of NucView™ 530 dye with dsDNA.
Figure 2. Detection of mitochondrial membrane potential, caspase activity, and apoptosis in MCF-7 cells. Cells were either untreated (A) or treated with staurosporine overnight to induce apoptosis (B), then stained with MitoView™ Blue, NucView™ 530 Caspase-3 Substrate, and CF™488A Annexin V for 30 minutes at 37oC in cell culture medium with no wash. Healthy cells show mitochondrial staining with MitoView Blue (cyan). Apoptotic cells lose MitoView™ Blue staining (cyan), and show NucView™ 520 staining of nuclei (red) and CF™488A Annexin V staining of cell membranes (green).
Figure 3. Jurkat cells were treated with DMSO (negative control, blue) or 0.1 uM, 0.25 uM, or 1 uM staurosporine (red) to induce apoptosis in the presence of NucView™ 530 and CF™405M Annexin V for four hours at 37oC before analysis on a BD LSR II flow cytometer. Staining with NucView™ 530 and CF™405M Annexin V increase with increasing concentration of staurosporine.
Cy dye is a registered trademark of GE Healthcare.
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