NucView® 488 Caspase-3 assay kit for Live Cells

Biotium products are distributed only in Singapore and Thailand.

NucView® 488 Caspase-3 substrate is a novel cell membrane-permeable fluorogenic caspase substrate designed for detecting caspase-3 activity within live cells in real time. Visit our Product Video page to see a video of NucView® 488 in action.

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 “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 intact cells without inhibiting caspase-3 activity. 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 intracellular caspase-3 activity and visualization of changes in nuclear morphology during apoptosis. The fluorescent staining produced in response to caspase-3 activity is compatible with subsequent fixation and permeabilization for immunostaining.

The kit contains NucView® 488 Caspase-3 substrate and a caspase-3 inhibitor Ac-DEVD-CHO sufficient for 100 flow cytometry or microplate assays (200 uL assay volume). The trial size kit contains sufficient reagents for 25 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. The kit provides a convenient tool for profiling apoptotic cell population based on caspase-3 activity using either fluorescence microscopy or flow cytometry.

Kit Components:

NucView® 488 Caspase-3 substrate, 0.2 mM in DMSO
Caspase-3 inhibitor Ac-DEVD-CHO, 2 mM in DMSO

Features:

Real time caspase-3 detection in intact cells: Detect caspase-3 activity within individual cells in a population.
Bi-functional: Allows visualization of caspase-3 activity and apoptotic nuclear morphology at the same time.
Simple & Fast: 15-minute homogenous assay with no wash required.
Versatile: For detection by flow cytometry, fluorescence microscopy, or fluorescence microplate reader using fluorescein detection settings.
Fixable: Compatible with standard fixation and permeabilization methods for subsequent immunofluorescence staining.

We also offer NucView® 488 Caspase-3 Substrate at 1 mM in DMSO (10402) or 1 mM in PBS (10403), for customers who wish to control the concentration of DMSO in their assay. Note that in cell types that are not sensitive to DMSO, the presence of DMSO during substrate incubation can enhance signal. See our full selection of NucView® substrates and kits.

NucView® enzyme substrate technology is covered by U.S. Patents.

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

Reference Publications

Smith, Graham S T et al.

Monitoring cleaved caspase-3 activity and apoptosis of immortalized oligodendroglial cells using live-cell imaging and cleaveable fluorogenic-dye substrates following potassium-induced membrane depolarization

Journal of visualized experiments : JoVE ,59 3422.

DOI: 10.3791/3422

Article Snippet: “We are grateful to Dr. Joan Boggs (Hospital for Sick Children, Toronto) for many helpful discussions and comments on this manuscript. We are grateful to Biotium for their generous gift of additional NucView 488 caspase-3.”

Guisier, Florian et al.

In- and ex-vivo molecular imaging of apoptosis to assess sensitivity of non-small cell lung cancer to EGFR inhibitors using probe-based confocal laser endomicroscopy

PloS one vol. 12,7 e0180576.

DOI: 10.1371/journal.pone.0180576

Article Snippet: “The NucView 488 Caspase-3 substrate (Biotium, San Francisco, California, USA), hereafter referred to as C3-NucView, is a fluorogenic dye that detects caspase 3 activity within intact cells, without interfering with the caspase 3 activity.”

Evelina Folkesson, Barbara Niederdorfer, VuTo Nakstad, LivThommesen, Geir Klinkenberg, Astrid Lægreid & Åsmund Flobak.

High‑throughput screening reveals higher synergistic efect of MEK inhibitor combinations in colon cancer spheroids

Scientifc Reports (2020) 10:20946

DOI: https://doi.org/10.1038/s41598-020-77501-4

Article Snippet: “Assay reagents used in screens were CellTiter-Glo 2.0 Assay (Promega), CellTiter-Glo 3D Cell Viability Assay (Promega), CellTox Green Cytotoxicity Assay17 (Promega) and NucView 488 Caspase-3 Substrate18 (Biotium).”

Pevná V, Máčajová M, Hovan A, Bánó G, Meta M, Bilčík B, Palková J, Huntošová V.

Spheroidal Model of SKBR3 and U87MG Cancer Cells for Live Imaging of Caspase-3 during Apoptosis Induced by Singlet Oxygen in Photodynamic Therapy

Biomedicines. 2022; 10(9):2141.

DOI: https://doi.org/10.3390/biomedicines10092141

Article Snippet: “Fluorescence was excited either with solid-state lasers at 405 nm, 488 nm, and 555 nm or with light from the mercury-tungsten lamp filtered with the filter cube FS10 (ZEISS, excitation BP 450–490, emission BP 515–565) for the NucView®488 caspase-3 substrate (biotium, Fremont, CA, USA)“

Ibarra Chacon, Andrea F.

Evaluation Of The Cytotoxyc Effects Of LMDF5-7 On A Human T-Cell Leukemia Cell Line

Open Access Theses & Dissertations. 3272.

DOI: https://scholarworks.utep.edu/open_etd/3272

Article Snippet: “The assay consists of a fluorogenic substrate which is cleaved by an active caspase-3 using NucView 488 caspase-3 substrate assay kit (Biotium, Hayward, CA), releasing a product that after binding to the DNA emits a bright green fluorescence signal.”