This kit provides two apoptosis markers, our novel NucView™ 488 caspase-3 substrate and Sulforhodamine 101 (Texas Red®) Annexin V, for profiling apoptotic cells and studying the relation of two important apoptosis events, caspase-3 activation and phosphatidylserine (PS) translocation. The rate of apoptosis varies from cell to cell even within the same population. As a result, various apoptotic events or markers accompanying the apoptotic process also occur differently among cells. Thus, it is important to be able to detect these apoptotic events on an individual cell basis.
Traditionally, caspase activity has been detected either using a membrane-impermeable fluorogenic enzyme substrate such as DEVD-R110, or a fluorescently-labeled inhibitor such as a FLICA reagent. In the former case, cell lysis is required, thus precluding the detection of caspase activity in live cells. In addition, such caspase assays measure only the average caspase activity of a highly heterogeneous cell population at a given time. In the latter case, although a FLICA reagent can enter live cells to detect caspase activity, only the initial fluorescent signal following the application of the reagent can truly reflect the enzyme activity or the state of the apoptotic cells because any detected signal after the initial “snapshot” will need to consider the potential interference of the inhibitor to the enzyme and the apoptotic cell itself.
Unlike conventional caspase assays, NucView™ 488 Caspase-3 substrate detects caspase-3 activity within individual whole cells in a non-interfering manner. The substrate consists of a fluorogenic DNA dye and a DEVD substrate moiety specific for caspase-3. The substrate, which is both non-fluorescent and nonfunctional as a DNA dye, rapidly crosses cell membranes to enter the cytoplasm, where it is cleaved by caspase-3 to form a high-affinity DNA dye that stains the nucleus bright green. Thus, the NucView™ 488 caspase-3 substrate is bi-functional, allowing detection of caspase-3 activity and visualization of apoptotic nuclear morphology. Also provided in the kit is Sulforhodamine 101 (Texas Red®) Annexin V, a red fluorescent probe for detecting phosphatidylserine (PS) translocation, another apoptosis marker.
The kit provides a convenient tool for simultaneous detection of caspase-3 activity and PS translocation and for studying the correlation of these two apoptotic events by fluorescence microscopy or flow cytometry. The fluorescence spectra of the cleaved substrate and sulforhodamine 101 are well separated by using the fluorescein filter set and Texas Red® filter set, respectively. Also see our Dual Apoptosis Assay with NucView™ 488 and CF™594 Annexin V, our improved version of this kit featuring CF™594 Annexin V, which is spectrally similar to Texas Red®, but with improved brightness and photostability. The kit contains reagents sufficient for 50 flow cytometry assays (200 uL assay volume).
The number of fluorescence microscopy assays that can be performed with the kit may vary based on the size of culture vessel and staining volume used.
- 1 vial (250 uL/vial); NucView™ 488 Caspase-3 substrate, 0.2 mM in DMSO
- 1 vial (250 uL/vial); Sulforhodamine 101 (Texas Red®) Annexin V
- 1 vial (15 mL/vial); 5X binding buffer
- 1 vial (100 uL/vial); Caspase-3 inhibitor Ac-DEVD-CHO, 2 mM in DMSO
- Multi-functional: NucView™ 488 caspase-3 substrate detecting caspase-3 activity and the morphological changes of cell nuclei, and Sulforhodamine 101 (Texas Red®) Annexin V detecting PS exposure on the cell surface.
- Simple & Fast: 15-30 minute staining.
- Versatile: Compatible with flow cytometry or fluorescence microscopy using fluorescein filter set for NucView™ 488 caspase-3 substrate and Texas Red® filter set for CF™594-annexin V, respectively.
See our full selection of NucView™ substrates and kits. NucView™ enzyme substrate technology is covered by U.S. patents. Texas Red® is a registered trademark of Molecular Probes.Compatible with the NucleoCounter® NC-3000™.