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Telephone:1-408-733-1055
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Amplite® Fluorimetric Caspase 3/7 Assay Kit *Blue Fluorescence*

Caspases play important roles in apoptosis and cell signaling. The activation of caspase-3 (CPP32/apopain) is important for the initiation of apoptosis. Caspase 3 is also identified as a drug-screening target. Caspase 3 has substrate selectivity for the peptide sequence Asp-Glu-Val-Asp (DEVD). This Amplite® Caspase-3 Assay Kit uses Ac-DEVD-AMC as fluorogenic indicator for assaying caspase-3 activity. AMC-derived caspase substrates are widely used for fluorimetric detection of various caspase activities. Cleavage of AMC peptides by caspases generates strongly fluorescent AMC that is monitored fluorimetrically at 440-460 nm with excitation of 340-350 nm. This kit can be used to continuously measure the activities of caspase-3 in cell extracts and purified enzyme preparations using a fluorescence microplate reader or fluorometer.

Example protocol

AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds (100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate)
  2. Add equal volume of Caspase 3/7 assay working solution
  3. Incubate at room temperature for 1 hour
  4. Monitor fluorescence intensity at Ex/Em = 350/450 nm

Important notes
Thaw Component A, B, C (and if desired, Component D) at room temperature before use.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

(Optional) Caspase 3/7 Inhibitor Ac-DEVD-CHO stock solution (1 mM):
Add 100 µL of DMSO (not provided) directly to the vial of Caspase 3/7 Inhibitor Ac-DEVD-CHO (Component D). This inhibitor can be used to confirm the correlation between fluorescence signal intensity and Caspase 3/7-like protease activities.

PREPARATION OF WORKING SOLUTION

Add 50 μL of 200X Caspase 3/7 Substrate stock solution (Component A) and 100 μL of 1M DTT solution (Component C) into 10 mL of Assay buffer (Component B) and mix well. Note: 50 μL of the 200X Caspase 3/7 Substrate stock solution is enough for 100 assays using a reaction volume of 100 μL per assay. Keep from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

SAMPLE EXPERIMENTAL PROTOCOL

  1. Treat cells by adding 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-plate) in PBS or desired buffer. For blank wells (medium without the cells), add the corresponding amount of compound buffer.

  2. Incubate the cell plate in a 37°C, 5% CO2 incubator for a desired period of time (4 - 6 hours for Jurkat cells treated with camptothecin) to induce apoptosis.

  3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Caspase 3/7 working solution.

  4. Incubate the plate at room temperature for at least 1 hour, protected from light. Note: If desired, add 1 µL of the 1 mM stock solution of the Caspase 3/7 Inhibitor Ac-DEVD-CHO to selected samples 10 minutes before adding the assay solution at room temperature to confirm the caspase 3/7-like protease activities.

  5. Centrifuge the cell plate (especially for the non-adherent cells) at 800 rpm for 2 minutes with brake off.

  6. Monitor the fluorescence increase at Ex/Em = 350/450 nm.

Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)
Amplite® Fluorimetric Caspase 3/7 Assay Kit *Green Fluorescence*50052280000
Amplite® Fluorimetric Caspase 3/7 Assay Kit *Red Fluorescence*532619-

Citations

View all 22 citations: Citation Explorer
Goji Berry Juice Prevents Tumor Necrosis Factor Alpha-Induced Xerostomia in Human Salivary Gland Cells
Authors: Takakura, Masatoshi and Mizutani, Ayano and Kudo, Mizuki and Ishikawa, Airi and Okamoto, Takuya and Fu, Tong Xuan and Kurimoto, Shin-ichiro and Koike, Yuka and Mishima, Kenji and Tanaka, Junichi and others,
Journal: Biological and Pharmaceutical Bulletin (2024): 138--144
Respiratory syncytial virus--approved mAb Palivizumab as ligand for anti-idiotype nanobody-based synthetic cytokine receptors
Authors: Ettich, Julia and Wittich, Christoph and Moll, Jens M and Behnke, Kristina and Floss, Doreen M and Reiners, Jens and Christmann, Andreas and Lang, Philipp A and Smits, Sander HJ and Kolmar, Harald and others,
Journal: The Journal of Biological Chemistry (2023)
Respiratory syncytial virus (RSV)-approved monoclonal antibody Palivizumab as ligand for anti-idiotype nanobody-based synthetic cytokine receptors
Authors: Ettich, Julia and Wittich, Christoph and Moll, Jens M and Behnke, Kristina and Floss, Doreen M and Reiners, Jens and Christmann, Andreas and Lang, Philipp A and Smits, Sander HJ and Kolmar, Harald and others,
Journal: Journal of Biological Chemistry (2023): 105270
Role of mitochondrial dysfunction in the pathogenesis of cisplatin-induced myotube atrophy
Authors: Matsumoto, Chinami and Sekine, Hitomi and Nahata, Miwa and Mogami, Sachiko and Ohbuchi, Katsuya and Fujitsuka, Naoki and Takeda, Hiroshi
Journal: Biological and Pharmaceutical Bulletin (2022): b22--00171
Combinatorial targeting of a chromatin complex comprising Dot1L, menin and the tyrosine kinase BAZ1B reveals a new therapeutic vulnerability of endocrine therapy-resistant breast cancer
Authors: Salvati, Annamaria and Melone, Viola and Sellitto, Assunta and Rizzo, Francesca and Tarallo, Roberta and Nyman, Tuula A and Giurato, Giorgio and Nassa, Giovanni and Weisz, Alessandro
Journal: Breast Cancer Research (2022): 1--23

References

View all 67 references: Citation Explorer
In vivo and in vitro sensitization of leukemic cells to adriamycin-induced apoptosis by pentoxifylline. Involvement of caspase cascades and IkappaBalpha phosphorylation
Authors: Lerma-Diaz JM, Hern and ez-Flores G, Dominguez-Rodriguez JR, Ortiz-Lazareno PC, Gomez-Contreras P, Cervantes-Munguia R, Scott-Algara D, Aguilar-Lemarroy A, Jave-Suarez LF, Bravo-Cuellar A.
Journal: Immunol Lett (2006): 149
Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe
Authors: Wu X, Simone J, Hewgill D, Siegel R, Lipsky PE, He L.
Journal: Cytometry A (2006): 477
Quantitative measurement of caspase-3 activity in a living starfish egg
Authors: Sakaue M, Motoyama Y, Yamamoto K, Shiba T, Teshima T, Chiba K.
Journal: Biochem Biophys Res Commun (2006): 878
Photoreceptor cell apoptosis induced by the 2-nitroimidazole radiosensitizer, CI-1010, is mediated by p53-linked activation of caspase-3
Authors: Miller TJ, Schneider RJ, Miller JA, Martin BP, Al-Ubaidi MR, Agarwal N, Dethloff LA, Philbert MA.
Journal: Neurotoxicology (2006): 44
Diallyl Trisulfide Induces Apoptosis of Human Gastric Cancer Cell Line MGC803 Through Caspase-3 Pathway.
Authors: Xiao XL, Peng J, Su Q, Xiang SL, Tang GH, Huang YS, Zhou XT.
Journal: Ai Zheng (2006): 1247
Page updated on November 20, 2024

Ordering information

Price
Unit size
Catalog Number13502
Quantity
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Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
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Custom sizeInquire
Technical SupportContact us
Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Spectral properties

Excitation (nm)

341

Emission (nm)

441

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200

Platform

Fluorescence microplate reader

Excitation350 nm
Emission450 nm
Cutoff420 nm
Recommended plateSolid black

Components

Detection of Caspase 3/7 activity in Jurkat cells with Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit. Jurkat cells were seeded on the same day at 80,000 cells/well/90 µL in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 1 µM of staurosporine for 4 hours, and with or without 10 µM of the caspase inhibitor AC-DEVD-CHO for 10 minutes. The caspase 3/7 assay solution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 350/450 nm (Cutoff = 420 nm).
Detection of Caspase 3/7 activity in Jurkat cells with Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit. Jurkat cells were seeded on the same day at 80,000 cells/well/90 µL in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 1 µM of staurosporine for 4 hours, and with or without 10 µM of the caspase inhibitor AC-DEVD-CHO for 10 minutes. The caspase 3/7 assay solution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 350/450 nm (Cutoff = 420 nm).
Detection of Caspase 3/7 activity in Jurkat cells with Cell Meter™ Caspase 3/7 Activity Apoptosis Assay Kit. Jurkat cells were seeded on the same day at 80,000 cells/well/90 µL in a Costar black wall/clear bottom 96-well plate. The cells were treated with or without 1 µM of staurosporine for 4 hours, and with or without 10 µM of the caspase inhibitor AC-DEVD-CHO for 10 minutes. The caspase 3/7 assay solution (100 µL/well) was added and incubated at room temperature for 1 hour. The fluorescence intensity was measured at Ex/Em = 350/450 nm (Cutoff = 420 nm).
<strong>Effect of TA chimeras on liposomal Dox-mediated activation of caspase 3/7.&nbsp;</strong>HeLa M (A) and BeWo (B) cells were incubated with Dox-loaded liposomes with or without incorporated TA proteins as indicated. The activity of caspase 3 and caspase 7 was measured after 48 hours of incubation. The values shown are expressed as a percentage of the value obtained using untreated cells. Error bars correspond to the standard deviation (n = 3) and the significance of the acquired values relative to protein-free Dox-loaded liposomes was determined by one-way ANOVA test (* indicates p &lt; 0.05). Source:&nbsp;<strong>Sialic acid-binding lectin from bullfrog eggs inhibits human malignant mesothelioma cell growth <em>in vitro</em> and <em>in vivo</em></strong> by Takeo Tatsuta et al., <em>PLOS</em>, Jan. 2018.
Effect of ONA on tumour progression in mouse models. As a murine ovarian cancer model, C57B6 mice were injected in the right ovary with iMOC cells and were administered ONA (20&thinsp;mg/kg), as shown in the schematic diagram (A). Most of the untreated C57B6 mice died from cancer metastasis by day 40. The survival time (B) and tumour weight (C, scale bar: 1&thinsp;cm) were evaluated. STAT3 activation (D, scale bar: 20&thinsp;&mu;m), caspase-3 activation (E, scale bar: 200&thinsp;&mu;m), and the infiltration of macrophages (F) in the tumour tissues were evaluated using immunostaining. The percentage of F4/80- and CD163-positive cells in Iba-1-positive macrophages was presented (F). Then, nude mice were injected in the intraperitoneal cavity with ES2 cells and were administered ONA (20&thinsp;mg/kg), as shown in the schematic diagram (G) followed by the determination of the survival rate (G). Most of the untreated nude mice died from cancer metastasis by day 20. Source: <strong>Onionin A inhibits ovarian cancer progression by suppressing cancer cell proliferation and the protumour function of macrophages </strong>by Tsuboki et al., <em>Scientific Reports</em>, &nbsp;July 2016.
Combined effect of ONA and anti-cancer drugs in EOC cells. EOC cells (SKOV3, ES2, and RMG1) were incubated with a combination of both the ineffective concentration of individual anti-cancer drugs (PTX, CBDCA, or CDDP) and ONA for 24&thinsp;hours, followed by the determination of cell proliferation using the WST-8 assay (A) and the ineffective concentration of each anti-cancer drug for each cell line was determined. In addition, the EOC cells were incubated with anti-cancer drugs and ONA for 4&thinsp;hours, followed by caspase-3 measurement (B). The data are presented as the mean&thinsp;&plusmn;&thinsp;SD. *p-value&thinsp;&lt;&thinsp;0.05, **p-value&thinsp;&lt;&thinsp;0.01 vs. control (without anti-cancer drug). In addition, each EOC cell line (SKOV3: C, ES2: D, and RMG1: E) was incubated with an ineffective concentration of each anti-cancer drug (PTX, CBDCA and CDDP) with or without ONA for 3&thinsp;hours, followed by the measurement of pSTAT3, STAT3 and &beta;-actin by Western blot analysis, as described in the Materials and Methods. Source: <strong>Onionin A inhibits ovarian cancer progression by suppressing cancer cell proliferation and the protumour function of macrophages </strong>by Tsuboki et al., <em>Scientific Reports</em>, &nbsp;July 2016.

Documents

pdfSafety data sheet
pdfProduct protocol
pdfCertificate of analysis
Amplite® Reagents and Assay Kits
Caspases

Alternative formats

Amplite® Fluorimetric Caspase 3/7 Assay Kit *Green Fluorescence*
Amplite® Fluorimetric Caspase 3/7 Assay Kit *Red Fluorescence*