This kit provides two apoptosis markers, our novel NucView^TM 488 caspase-3 substrate and sulforhodamine 101-annexin V (Texas Red^TM-annexin V), for profiling apoptotic cell population and studying the relation of two important apoptosis events, caspase-3 activation and phosphatidylserine (PS) translocation.

The rate of apoptosis typically 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 "snap shot" will need to consider the potential interference of the inhibitor to the enzyme and the apoptotic cell itself.

Different from the conventional caspase assays, NucView^TM 488 Caspase-3 substrate detects caspase-3 activity within individual whole cells in a noninterfering manner. The substrate consists of a fluorogenic DNA dye and a DEVD substrate moiety specific for caspase-3. The substrate, which is both nonfluorescent and nonfunctional as a DNA dye, rapidly crosses cell membrane to enter the cell cytoplasm, where it is cleaved by caspase-3 to form a high-affinity DNA dye. The released DNA dye migrates to the cell nucleus to stain the nucleus bright green. Thus, the NucView^TM 488 caspase-3 substrate is bi-functional, being able to detect both intracellular caspase-3 and at the same time stain the cell nucleus, which is known to undergo morphological change during the apoptosis process. The fluorescent staining produced in response to caspase-3 activity is fixable via standard fixation method, thus facilitating any subsequent immunostaining study.

Also provided in the kit is sulforhodamine 101-annexin V, a red fluorescent probe for detecting phosphatidylserine (PS) translocation, which is another apoptosis marker. The kit provides a convenient tool for detecting both caspase-3 activity and PS translocation at the same time and for studying the correlation of these two apoptotic events by either 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^TM filter set, respectively.

• 1 vial (250 uL/vial); NucView^TM 488 Caspase-3 substrate, 0.2 mM in DMSO
• 1 vial (250 uL/vial); Sulforhodamine 101-annexin V (Texas Red^TM-annexin V), in TE buffer containing 0.1% BSA and 0.1% NaN₃, pH 7.5
• 1 vial (10 mL/vial); Annexin V binding buffer
• 1 vial (20 uL/vial); Caspase-3 inhibitor Ac-DEVD-CHO, 2 mM in DMSO

• Multiple Detections: NucView^TM 488 caspase-3 substrate detecting caspase-3 activity and the morphological changes of cell nuclei, and Sulforhodamine 101-annexin V detecting PS translocation on cell membrane.
• Simple & Fast: requiring only a 15-minute incubation time without washing for caspase-3-positive cells to be reliably detected.
• Versatile: compatible with either flow cytometry, or fluorescence microscopy using fluorescein filter set for NucView^TM 488 caspase-3 substrate and Texas Red filter set for Sulforhodamine 101-annexin V, respectively.