Cell biology 

This kit provides two apoptosis markers, NucView™ 488 Caspase-3 Substrate and Annexin V conjugated with a dye, for profiling apoptotic cells and studying the relation of two important apoptosis events, caspase-3 activation and phosphatidylserine (PS) translocation.

Annexin V conjugation for the second marker is available with the following dyes: Sulforhodamine 101 (Texas Red) Annexin V, CF594 Annexin V, and CF640R Annexin V.

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.

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.