This kit provides two apoptosis markers, our novel NucView™488 caspase-3 substrate and CF™640R 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 CF™640R Annexin V, a far-red fluorescent probe for detecting phosphatidylserine (PS) translocation, another apoptosis marker. CF™640R is spectrally similar to Cy®5, but with brighter fluorescence and excellent photostability. 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 CF™640R are well separated by using the fluorescein filter set and Cy®5 filter set, respectively. 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. Kit Components:
- 1 vial (250 uL/vial); NucView™488 Caspase-3 substrate, 0.2 mM in DMSO
- 1 vial (250 uL/vial); CF™640R 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 CF™640R 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 Cy®5 filter set for CF™640R Annexin V, respectively.
Biotium also offers a dual apoptosis assay that combines NucView 488 and deep-red fluorescent CF594 Annexin V, which is spectrally similar to Texas Red (catalog no. 30067). NucView™ enzyme substrate technology is covered by U.S. Patent Nos. 8,092,784 and 8,586,325. Texas Red is a registered trademark of Molecular Probes; Cy Dye is a registered trademark of GE Healthcare.Compatible with the NucleoCounter® NC-3000™.