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NucView® Caspase-3 Enzyme Substrates

Fluorescent caspase-3/7 substrates for detecting apoptosis in intact cells by confocal microscopy, flow cytometry, or live cell imaging.

Dye
Size
Catalog #
price
Qty
10 uL (1 mM in DMSO)
100 uL (1 mM in DMSO)
10 uL (1 mM in PBS)
100 uL (1 mM in PBS)
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Product Description

NucView® Caspase-3 Substrates are a convenient tool for detecting apoptosis in intact cells based on caspase-3/7 activity using confocal microscopy, flow cytometry, or live cell imaging.

  • Rapid, no-wash, endpoint or real-time assays
  • Non-toxic, allowing multi-day experiments to be performed
  • Available in green, blue, or orange fluorescence
  • For flow cytometry, microscopy or live cell imaging systems
  • Dual detection of caspase activity and nuclear morphology
  • Formaldehyde fixable

About NucView®

NucView® substrates consist of a fluorogenic DNA dye coupled to the caspase-3/7 DEVD recognition sequence. The substrate, which is initially non-fluorescent, penetrates the plasma membrane and enters the cytoplasm. In apoptotic cells, caspase-3/7 cleaves the substrate, releasing the high-affinity DNA dye, which migrates to the cell nucleus and stains DNA with fluorescence. Thus, NucView caspase-3 substrates are bifunctional, allowing detection of caspase-3/7 activity and visualization of morphological changes in the nucleus during apoptosis.

In contrast to other fluorogenic caspase substrates or fluorescent caspase inhibitor based (FLICA) assays, NucView® caspase-3 substrates can be used to detect caspase-3/7 activity within individual intact cells without inhibiting apoptosis progression. Staining is compatible with subsequent fixation and permeabilization for immunostaining.

NucView® substrates are offered as solutions in DMSO or phosphate-buffered saline (PBS). The substrates in PBS are formulated for use in cells that are sensitive to DMSO toxicity. In non-DMSO sensitive cell types, adding DMSO during the substrate incubation may enhance NucView® staining.

Download our E-Book: Visualizing Apoptosis as it Happens.

To learn about the advantages of monitoring apoptosis using NucView® caspase-3 substrates, visit the NucView® Technology Page.

NucView® Caspase-3 Substrates and Kits

Catalog No.

Features

NucView® 405 Caspase-3 Substrate, 1 mM in DMSO10405Blue fluorescence for flow cytometry in the Pacific Blue® channel or microscopy with the 405 nm laser
NucView® 405 Caspase-3 Substrate, 1 mM in PBS10407NucView® 405 substrate in PBS, for DMSO-sensitive cell types
NucView® 488 Caspase-3 Substrate, 1 mM in DMSO10402Green fluorescent substrate validated in more than 100 cell types and 200 publications
NucView® 488 Caspase-3 Substrate, 1 mM in PBS10403NucView® 488 substrate in PBS, for DMSO-sensitive cell types
NucView® 530 Caspase-3 Substrate, 1 mM in DMSO10406Orange fluorescence for microscopy in the Cy®3 channel or flow cytometry in the R-PE channel
NucView® 530 Caspase-3 Substrate, 1 mM in PBS10408NucView® 530 substrate in PBS, for DMSO-sensitive cell types
NucView® 488 and MitoView™ 633 Apotosis Detection Kit30062NucView® 488 and far-red fluorescent MitoView™ 633 for the Cy®5 channel
NucView® 488 and RedDot™ 2 Apoptosis & Necrosis Kit30072NucView® 488 and far-red dead cell DNA dye RedDot™ 2 for the Cy®5 channel
Dual Apoptosis Assay with NucView® 488 and CF®594 Annexin V30067NucView® 488 and red fluorescent Annexin V apoptosis probe
Dual Apoptosis Assay with NucView® 488 and CF®640R Annexin V30073NucView® 488 and far-red fluorescent Annexin V apoptosis probe

Choose from Blue, Green, or Orange Fluorescence

NucView® 488

NucView® 488 Caspase-3 Substrate stains apoptotic cell nuclei with green fluorescence, for detection in the FITC channel in fluorescence microscopy or flow cytometry. The dye is excited by the 488 nm laser line. NucView® 488 can be used for multi-color imaging with blue and far-red fluorescent probes. NucView® 488 is the original NucView® probe, and has been validated in over 200 publications and more than 100 cell types.

NucView® 405

NucView® 405  Caspase-3 Substrate stains apoptotic cell nuclei with blue fluorescence, for detection in the DAPI channel by confocal microscopy, or by flow cytometry in the Pacific Blue® channel. The dye is excited by the 405 nm laser line. NucView® 405 is ideal for caspase-3 detection in multi-color applications for researchers who wish to reserve the green fluorescence channel for other detection reagents.

Note: NucView® 405 signal may be difficult to observe using epifluorescence microscopy. Confocal microscopy using 405 nm laser excitation is recommended for imaging.

NucView® 530

NucView® 530 Caspase-3 Substrate stains apoptotic cell nuclei with orange fluorescence, for detection in the Cy®3 channel by fluorescence microscopy, or the PE channel by flow cytometry. The dye is excited by the 488 nm laser line. NucView® 530 can be used for multi-color imaging with blue, green, or far-red fluorescent probes.

Note: When excited by the 488 nm laser line, NucView® 530 also fluoresces in the FITC channel, and therefore cannot be analyzed together with green probes by flow cytometry.

NucView is a registered trademark of Biotium. NucView enzyme substrate technology is covered by U.S. patents.
References

Product Attributes

Size
10 uL (1 mM in DMSO), 100 uL (1 mM in DMSO), 10 uL (1 mM in PBS), 100 uL (1 mM in PBS)
Dye
NucView® 405, NucView® 488, NucView® 530
Apoptosis/viability marker
Caspase
For live or fixed cells
For live/intact cells
Assay type/options
Endpoint assay, Homogeneous assay, Long term staining (24-72h), No-wash staining, Real-time imaging
Detection method/readout
Fluorescence microscopy, Live cell imaging, Flow cytometry
Substrate specificity
Caspases
Colors
Blue, Green, Orange
Fixation options
Fix after staining (formaldehyde), Permeabilize after staining
Excitation/Emission
429/469 nm (with DNA), 500/530 nm (with DNA), 528/563 nm (with DNA)
Storage Conditions
Store at 2 to 8 °C, Store at -10 to -35 °C

Documents, Protocols, SDS and COA

FAQs

NucView® Caspase-3 Enzyme Substrates

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:

  • Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
  • Acetoxymethyl esters (AM esters), such as our membrane-permeable ion indicator dyes
  • Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF® Dye succinimidyl ester stability

Succinimidyl esters (SE) are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Many commercially available fluorescent dyes used for life science research are heavily sulfonated dyes which makes them particularly hygroscopic, worsening the hydrolysis problem. In addition, for several commercially available SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while the SE group in all of Biotium’s CF® Dyes is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF® Dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

The NucView® caspase-3 substrates are activity-dependent i.e. require active caspase-3 enzyme. In dead, fixed and preserved cells or tissue sections, there are no active caspases and hence these substrates cannot be used. We also offer TUNEL assay kits that are suitable for apoptosis detection in fixed cells and tissues. The kits employ dUTPs conjugated to our exceptionally bright and photostable CF® dyes for single-step fluorescent TUNEL labeling of DNA strand breaks, a hallmark of apoptotic cells, and are suitable for analysis by fluorescence microscopy or flow cytometry.

The NucView® 488 Assay Kit for Live Cells (30029) contains NucView® 488 substrate at 0.2 mM in DMSO and caspase-3 inhibitor Ac-DEVD-CHO. The final DMSO concentration in the kit assay can be fairly high, which is undesirable for sensitive cell types. Because of this, we offer NucView® 488 Caspase-3 Substrate at 1 mM in DMSO (10402) and at 1 mM in PBS (10403), so customers can control the amount of DMSO in their assay. Note that in non-DMSO sensitive cell types, adding DMSO during the substrate incubation can enhance NucView® staining.

We also offer blue fluorogenic NucView® 405 in DMSO (10405) and PBS (10407), and orange fluorogenic NucView® 530 in DMSO (10406) and PBS (10408).

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