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NucView™ Caspase-3 Substrates

Features:

    • Real-time detection of caspase-3/7 activity in intact cells
    • Homogenous, no wash 15-30 minute assay, or long-term real-time monitoring of caspase activity
    • Fixable nuclear staining
    • NucView™ 488: Green fluorescence detection for flow cytometry, microscopy, or microplate reader
    • NucView™ 405: Blue fluorescence detection for flow cytometry or microscopy using the 405 nm laser line
    • NucView™ 530: Orange fluorescence detection for microscopy or flow cytometry

Principle of NucView Caspase-3 Substrate Detection.


New video from Essen Bioscience featuring NucView™ 488 Caspase 3 Substrate

Cancer Cell Apoptosis Imaged by Incucyte ZOOM™ with NucView488 Caspase-3 substrate

See NucView™ 488 Caspase-3 Enzyme Substrate in action in this video provided by Essen Bioscience using their IncuCyte ZOOM™ live cell imaging system. Cells from the HT-1080 human fibroscarcoma-derived cell line were treated with 150 nM camptothecin in the presence of 5 uM NucView™ 488 Caspase-3 Substrate. The cells were imaged every 30 minutes for 24 hours, during which apoptosis can be observed by changes in cellular morphology and green fluorescence that is emitted upon DNA binding from the DNA binding NucView™ 488 free dye released by the activity of Caspase-3 upon the substrate.


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NucView™ product list


NucView™ Caspase-3 Substrates

DEVD-NucView™ Caspase-3 Substrates are a novel fluorogenic substrate developed by Biotium to follow caspase-3 activity in intact cells in real-time. We have invented a whole new concept to design fluorogenic enzyme substrates by attaching an enzyme substrate moiety to a functional dye, thereby making the functional dye nonfunctional. Upon enzymatic cleavage of the substrate, the dye restores its functionality and thus becomes capable of binding to its target to emit fluorescence.

In the case of NucView Caspase-3 Substrates, the caspase-3 substrate peptide DEVD is attached to a DNA-binding dye, making the dye unable to bind to DNA and thus non-fluorescent. The substrate enters the cell cytoplasm where it is cleaved by caspase-3 to release the high-affinity fluorogenic DNA dye, which migrates to the cell nucleus to stain the nucleus. Therefore, the bi-functional substrate allows one not only to detect caspase-3 within an apoptotic cell but also to visualize apopototic nuclear morphology. Unlike fluorescently-labeled caspase inhibitor assays (FLICA) that use irreversible inhibitors to label active caspases, NucView Caspase-3 Substrates do not interfere with caspase activity, allowing monitoring of caspase activity in real time.

Green fluorogenic NucView™ 488 Caspase-3 Substrate can be detected in the FITC channel by fluorescence microscopy, flow cytometry, or fluorescence microplate reader. Detection of apoptosis using NucView™ 488 Caspase-3 Substrate has been reported in more than 90 different primary and immortalized cells types, including 3-D cultures, in more than 150 publications.

Now, in addition to our green fluorogenic NucView™ 488 Caspase-3 Substrate, Biotium offers the new blue NucView™ 405 and orange NucView™ 530 fluorogenic caspase-3 substrates. NucView™ 405 dye is excited by the 405 nm laser line for detection by confocal microscopy in the DAPI channel (Figure 2), or by flow cytometry in the Pacific Blue® channel (Figure 3),  for multi-color applications in which the green fluorescence channel is reserved for other detection reagents. NucView™ 530 (excitation/emission 528/563 nm) can be detected by microscopy or flow cytometry in the Cy®3 or R-PE channels.

NucView™ Caspase-3 Substrates are available as a stand-alone substrates, or in kits with other fluorescent apoptosis and necrosis probes for studying the temporal and spatial relations among the different events by fluorescence microscopy or flow cytometry. See NucView™ products…

Time course of caspase-3 detection using NucView™ 488 Caspase-3 Substrate in Jurkat cells treated with staurosporine to induce apoptosis, analyzed in the FL1 channel of a BD FACSCalibur flow cytometer.
Apoptotic Jurkat cell stained with NucView™ 488 Caspase-3 Substrate (green) and CF647 Annexin V (magenta).

Flow cytometry analysis of apoptosis using NucView 405 caspase-3 substrate. Jurkat cells were incubated with staurosporine for the times indicated to induce apoptosis, then stained with 1 uM NucView 405 caspase-3 substrate for 30 minutes at room temperature. Fluorescence was analyzed using a BD LSR II flow cytometer in the Pacific Blue® channel (405 nm excitation, 450/50 nm emission filter). The graph shows the percentage of NucView-positive cells at each timepoint of staurosporine treatment.
Time course of caspase-3 detection with NucView™ 405 Caspase-3 Substrate in Jurkat cells treated with staurosporine to induce apoptosis, analyzed in the Pacific Blue® channel of a BD LSRII flow cytometer (405 nm excitation, 450/50 nm emission filter).
MitoView Blue, NucView 530, CF488A Annexin MCF7
Detection of caspase activity with NucView™ 530 (red), mitochondrial membrane potential with MitoView™ Blue (cyan), CF™488A Annexin V staining (green) in MCF-7 cells either left untreated (A) or treated with staurosporine overnight to induce apoptosis (B).