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Dual Apoptosis Assay with NucView 488 Caspase-3 Substrate & Annexin V

This kit provides two apoptosis markers, our novel NucView™ 488 Caspase-3 Substrate and Annexin V conjugated with your choice of red or far-red dye. The kit is used for studying two important apoptosis events, caspase-3 activation and phosphatidylserine (PS) translocation.

Apoptotic Jurkat cell stained with NucView 488 (green) and CF647 Annexin V (magenta).
Catalog #

30030, 30067, - 30073View allHide

Size

50 assays

Conjugation

CF594, CF640R, Sulforhodamine 101 (texas red)

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Product Description

This kit provides two apoptosis markers, our novel 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, CF™594 Annexin V, and CF™640R 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.

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.

References

Cen, et al. DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells. The FASEB Journal published online February 8, 2008.
Download a list of NucView references: Validated cell lines for NucView 488 with references

Download a full list of references: Validated cell lines for NucView 488 with references

1. PLoS Genet (2014), doi:10.1371/journal.pgen.1004594

2. Cancer Gene Therapy (2016), doi:10.1038/cgt.2016.18

 

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Support & Faq

NucView™ Caspase 3 Enzyme Substrates

The substrate is very stable. Some users have reported performing time course assays with NucView™ 488 Caspase-3 Substrate for 4-5 days.

Category: NucView™ Caspase 3 Enzyme Substrates

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NucView™ Caspase-3 Substrates can be added to the cells at the start of the experiment or at the end. A major advantage of NucView™ Caspase-3 Substrates compared to other apoptosis assays is that it can be used to monitor capase-3 activity in real time.

Category: NucView™ Caspase 3 Enzyme Substrates

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Yes. NucView™ Caspase-3 Substrates are compatible with flow cytometers and other instruments with the appropriate excitation and emission settings.

Category: NucView™ Caspase 3 Enzyme Substrates

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Apoptosis, Necrosis, and Cell Viability Kits

MTT and XTT are colorimetric based assays, while resazurin can be measured using colorimetric or fluorescence detection. MTT is not a soluble product, so the cells must be lysed to solubilize the formazan salt before absorbance can be measured. XTT and resazurin do not require cell lysis, allowing kinetic monitoring of the same samples at different timepoints.

Category: Apoptosis, Necrosis, and Cell Viability Kits

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Check to make sure your cell densities are in the linear range of the cell viability assay. Too few cells may fall below the limit of detection, and too many cells may saturate the OD. Perform an initial experiment to check the best linear range for your cell type and experimental set-up. You may need to vary the cell density assay incubation time. The kit protocols provide general guidelines and may need to be optimized for your experimental system.

Category: Apoptosis, Necrosis, and Cell Viability Kits

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Resazurin is reduced by cells to the fluorescent product resorufin. Resorufin can be reduced further to a non-fluorescent compound. Therefore the densest wells may have lowest fluorescence due to over-reduction of the substrate. Please see the product information sheet for more details. The kit protocol provides general guidelines and may need to be optimized empirically for your experimental system. You may need to vary cell density or assay incubation time to ensure that your samples fall in the linear range of the kit.

Category: Apoptosis, Necrosis, and Cell Viability Kits

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Shipping/Shelf-life

Shipment Method: Shipping and handling methods will be assessed and calculated at time of shipment based upon item(s) storage temperature conditions. Expedited shipment may be requested at time of checkout. Please note that products with recommended storage at 4°C or -20°C may ship at ambient temperature. This will not affect product performance. When you receive the product, place it under the recommended storage conditions.