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NUCVIEW® CASPASE-3 SUBSTRATES

The Only Caspase Substrates for Real-Time Apoptosis Detection

NucView® caspase-3 substrates are Biotium’s patented fluorogenic substrates designed for real-time apoptosis detection in live cells.

Real-time apoptosis detection
Substrate doesn't interfere with apoptosis progression.
For flow cytometry, microscopy, or live cell imaging.
Fast, simple, and fixable
Homogeneous, no wash 15-30 minute assay.
Formaldehyde-fixable nuclear staining.
Three color options
Blue fluorescent NucView® 405
Green fluorescent NucView® 488
Orange fluorescent NucView® 530

HOW IT WORKS

Principle of NucView® substrate technology. The substrate is non-fluorescent and does not bind DNA. After enzyme cleavage, the high-affinity DNA dye is released to bind DNA and become fluorescent.

NucView® caspase-3 substrates are based on novel fluorogenic DNA dyes that have been coupled to the caspase-3/7 recognition sequence (DEVD). When introduced to cells, the substrate is initially non-fluorescent and is able to penetrate the plasma membrane and enter the cytoplasm. During apoptosis, caspase-3/7 cleaves the substrate and releases the high-affinity DNA dye which leads to nuclear fluorescent staining. Consequently, 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 that which inhibit caspase activity, NucView® caspase-3 substrates can be used to detect caspase-3/7 activity in live cells without inhibiting apoptosis progression. Staining is also compatible with subsequent fixation and permeabilization for downstream immunostaining.

Features

  • Dual detection of caspase activity and nuclear morphology
  • Rapid, no-wash, endpoint or real-time assays
  • Non-toxic, allowing for multi-day experiments
  • For flow cytometry, microscopy, or live cell imaging systems
  • Available as stand-alone substrates or in kits paired with other apoptosis or necrosis probes
Click to view GIF animation. HeLa cells were stained with NucView® 488 Caspase-3 Substrate (2 uM), MitoView™ 633 mitochondrial potential dye (50 nM), and Hoechst 33342 blue nuclear stain (1 ug/mL), then treated with 1 uM staurosporine to induce apoptosis.

Available in blue, green, or red fluorescence

NucView® 405 Caspase-3 Substrate, 1 mM in PBS
Apoptotic HeLa cells stained with NucView® 405 Caspase-3 Substrate (blue) and CF®488A Annexin V (green).
Apoptotic HeLa cells stained with NucView® 488 Caspase-3 Substrate (green) and CF®594 Annexin V (red).
NucView 530 CF488A Annexin
Apoptotic MCF-7 cells stained with NucView® 530 Caspase-3 Substrate (red) and CF®488A Annexin V (green).

THE GOLD STANDARD FOR MONITORING APOPTOSIS

NucView® 488 is validated in more than 100 cell types and 200 publications

Highlighted Citation: NucView® 488 was used in a T cell-mediated tumor cell killing assay by Lim et al. in a study investigating the role of inflammation on PD-L1 stabilization. Results show T-cell mediated killing of PD-L1 expressing BT549 ductal carcinoma cells was no longer suppressed when treated with neutralizing TNF-α antibody. In addition, PD-L1 depleted cells did not respond to either cytokine enriched macrophage-conditioned medium (MP) or TNF-α antibody, suggesting that PD-L1 expression is required for TNF- α-mediated suppression of T cell activity.

See the full list of references here.

T cell-meditated tumor cell killing assay in PD-L1 knockout (PD-L1 KO) BT549 cells. Representative phase, red fluorescent (nuclear restricted RFP), and green fluorescent (NucView 488 caspase-3/7 substrate) merged images of activated T cell co-cultures in the presence of caspase-3/7 substrate at 96 hr are shown. T cells were activated with CD3 antibody (100 ng/mL) and IL-2 (10 ng/mL). Green fluorescent cells were counted as dead cells. The quantitative ratio of dead cells is shown in the bar graph on the right. Scale bar, 100 um. Credit: Lim et al. doi.org/10.1016/j.ccell.2016.10.010

DUAL DETECTION KITS AVAILABLE

Planning on using NucView® Caspase-3 Substrates with other markers?

See our dual detection apoptosis kits which pair NucView® 488 with either Annexin V conjugates, MitoView™ 633, or RedDot™2.

  • Dual Apoptosis Kit: NucView® 488 and Annexin V labeled with red or far-red dyes for co-detection of two apoptotic events, caspase cleavage and phosphatidylserine (PS) translocation.
  • Dual Apoptosis Kit: NucView® 488 and MitoView™ 633 for co-detection of two apoptotic events, caspase cleavage and loss of mitochondrial membrane potential.
  • Apoptosis/Necrosis Kit: NucView® 488 and RedDot™2 for concurrent measurement of caspase cleavage (apoptosis) and loss of membrane integrity (necrosis).

MCF-7 cells were left untreated or treated with staurosporine to induce apoptosis, then with 40 nM MitoView™ Blue, NucView® 520 Caspase-3 Substrate, and CF® 488A Annexin V. Healthy cells show mitochondrial staining with MitoView™ Blue (cyan), while apoptotic cells show NucView® 520 staining of nuclei (red) and CF® 488A Annexin V staining of cell membranes (green).

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

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

From: $362 Sizes: 50 assaysCatalog #: 30030, 30067, - 30073View allHide

NucView® Caspase-3 Enzyme Substrates

From: $67 Sizes: 10 uL (1 mM in DMSO), 100 uL (1 mM in DMSO), 10 uL (1 mM in PBS), 100 uL (1 mM in PBS)Catalog #: 10405-T, 10405, 10407-T, 10407, 10402-T, 10402, 10403-T, 10403, 10406-T, 10406, 10408-T, - 10408View allHide

NucView® 488 & MitoView™ 633 Apoptosis Assay Kit

From: $351 Sizes: 100 assaysCatalog #: 30062

NucView® 488 & RedDot™ 2 Apoptosis & Necrosis Kit

From: $308 Sizes: 100 assaysCatalog #: 30072

NucView® 488 Caspase-3 Assay Kit for Live Cells

From: $98 Sizes: Trial size (25 assays), 100 assaysCatalog #: 30029, - 30029-TView allHide

 

Faq

NucView® Caspase-3 Enzyme Substrates

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature or with an ice pack. These products may thaw without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Category: CF® Dyes, DNA Quantitation Kits, Luciferase Assays, Mix-n-Stain™ Antibody Labeling Kits, NucView® Caspase-3 Enzyme Substrates, Product shipping, storage, shelf life, & solubility

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Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, 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. One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.  

Category: Apoptosis, Necrosis, and Cell Viability Kits, CF® Dyes, DNA Quantitation Kits, GelRed® and GelGreen® Nucleic Acid Gel Stains, Luciferase Assays, Mix-n-Stain™ Antibody Labeling Kits, NucView® Caspase-3 Enzyme Substrates, Product shipping, storage, shelf life, & solubility

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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 are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDyes® are particularly hygroscopic, worsening the hydrolysis problem. For example, the percent of active Alexa Fluor® 488 succinimidyl ester (SE) could be well below 50% by the time of application (according to the manufacturer’s product datasheet). In a number of Alexa Fluor® SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while in all of Biotium’s CF™ dyes the SE group 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.

Category: CF® Dyes, DNA Quantitation Kits, Luciferase Assays, Mix-n-Stain™ Antibody Labeling Kits, NucView® Caspase-3 Enzyme Substrates, Product shipping, storage, shelf life, & solubility

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