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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 24 citations: Citation Explorer
BRPF1 inhibition reduces migration and invasion of metastatic ovarian cancer cells, representing a potential therapeutic target
Authors: Alexandrova, Elena and Smal, Marharyta and Di Rosa, Domenico and Brancaccio, Rosario Nicola and Parisi, Roberto and Russo, Fabio and Tarallo, Roberta and Nassa, Giovanni and Giurato, Giorgio and Weisz, Alessandro and others,
Journal: Scientific Reports (2025): 7602
An engineered Palivizumab IgG2 subclass for synthetic gp130 and fas mediated signaling
Authors: Wittich, Christoph and Ettich, Julia and Hertell, Marcel and Roy, Biswadeep G and Xu, Haifeng C and Floss, Doreen M and Lang, Philipp A and Scheller, J{\"u}rgen
Journal: Journal of Biological Chemistry (2025): 108205
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

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 March 11, 2025

Ordering information

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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*