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Amplite® Colorimetric Caspase 3/7 Assay Kit *Yellow Color*
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Application notes
Caspases play important roles in apoptosis and cell signaling. The activation of Caspase 3/7 (CPP32/apopain) is important for the initiation of apoptosis. Caspase 3/7 is also identified as a drug-screening target. Caspase inhibitors have anti-cancer and other pharmalogical potentials. It has been proven that Caspase 3/7 has substrate selectivity for the peptide sequence Asp-Glu-Val-Asp (DEVD). Our Amplite® Colorimetric Caspase 3/7 Assay Kit uses (Z-DEVD)2R110 as the chromogenic indicator for assaying caspase 3/7 activity. R110 peptide sustrates are colorless. Cleavage of R110 peptides by caspases generates R110, a yellow color dye that can be monitored at 490-520 nm. The increase in the absorbance of caspase-induced R110 is proportional to the activities of caspases. This kit can be used to continuously measure the activities of caspase 3/7 in cell extracts and purified enzyme preparations with an absorbance microplate reader with much higher sensitivity than the other commercial kits that use DEVD-pNA peptide.
Example protocol
AT A GLANCE
Protocol Summary
- Prepare cells with test compounds (100 µL/well/96-well plate or 25 µL/well/384-well plate)
- Add equal volume of Caspase 3/7 working solution (100 µL/well/96-well plate or 25 µL/well/384-well plate)
- Incubate at room temperature for 1 - 2 hours
- Monitor absorbance at 490 nm
CELL PREPARATION
For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html
PREPARATION OF WORKING SOLUTION
Add 50 μL of 200X Caspase 3/7 Substrate stock solution (Component A) into 10 mL Assay Buffer (Component B), and mix well to make Caspase 3/7 working solution. Note: This Caspase 3/7 working solution is enough for 100 assays using a reaction volume of 100 μL per assay. Before opening the vial, do brief centrifuge to accumulate the stock solution to the bottom of the tube.
SAMPLE EXPERIMENTAL PROTOCOL
- Treat cells with 10 µL of 10X test compounds (for a 96-well plate) or 5 µL of 5X test compound (for a 384-well plate) in PBS or desired buffer. For blank wells (medium without the cells), add the corresponding amount of compound buffer.
- Incubate the cell plates in an incubator for a desired period of time to induce apoptosis. Note: We treated Jurkat cells with staurosporine (SS) for 4 hours at 37°C to induce cell apoptosis. See Figure 1 for details.
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of Caspase 3/7 working solution.
- Incubate the plate at room temperature for at least 1 hour, protected from light.
- Centrifuge cell plates at 800 rpm for 2 minutes with brake off.
- Monitor the absorbance increase with an absorbance plate reader at OD =490 nm.
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) |
| Amplite® Fluorimetric Caspase 3/7 Assay Kit *Blue Fluorescence* | 341 | 441 |
| Amplite® Fluorimetric Caspase 3/7 Assay Kit *Green Fluorescence* | 500 | 522 |
| Amplite® Fluorimetric Caspase 3/7 Assay Kit *Red Fluorescence* | 532 | 619 |
Citations
View all 10 citations: Citation Explorer
Linking pesticide exposure to neurodegenerative diseases: An in vitro investigation with human neuroblastoma cells
Authors: Alehashem, Maryam and Alcaraz, Alper James and Hogan, Natacha and Weber, Lynn and Siciliano, SD and Hecker, M
Journal: Science of The Total Environment (2024): 173041
Authors: Alehashem, Maryam and Alcaraz, Alper James and Hogan, Natacha and Weber, Lynn and Siciliano, SD and Hecker, M
Journal: Science of The Total Environment (2024): 173041
Chrysin Mediates the Induction of Apoptosis in Breast Cancer Cells via the Inhibition of the WNT/$\beta$-Catenin Signaling Pathway
Authors: {\c{C}}etinkaya, S{\"u}meyra
Journal: (2023)
Authors: {\c{C}}etinkaya, S{\"u}meyra
Journal: (2023)
Folate-Decorated Cross-Linked Cytochrome c Nanoparticles for Active Targeting of Non-Small Cell Lung Carcinoma (NSCLC)
Authors: Dominguez-Martinez, Irivette and Joaquin-Ovalle, Freisa and Ferrer-Acosta, Yancy and Griebenow, Kai H
Journal: Pharmaceutics (2022): 490
Authors: Dominguez-Martinez, Irivette and Joaquin-Ovalle, Freisa and Ferrer-Acosta, Yancy and Griebenow, Kai H
Journal: Pharmaceutics (2022): 490
Cross-Linked Cytochrome c-Based Nanoparticles for Targeted and Controlled Cancer Therapy
Authors: Martinez, Irivette Dominguez
Journal: (2022)
Authors: Martinez, Irivette Dominguez
Journal: (2022)
High STMN1 level is associated with chemo-resistance and poor prognosis in gastric cancer patients
Authors: Bai, Tuya and Yokobori, Takehiko and Altan, Bolag and Ide, Munenori and Mochiki, Erito and Yanai, Mitsuhiro and Kimura, Akiharu and Kogure, Norimichi and Yanoma, Toru and Suzuki, Masaki and others, undefined
Journal: British Journal of Cancer (2017)
Authors: Bai, Tuya and Yokobori, Takehiko and Altan, Bolag and Ide, Munenori and Mochiki, Erito and Yanai, Mitsuhiro and Kimura, Akiharu and Kogure, Norimichi and Yanoma, Toru and Suzuki, Masaki and others, undefined
Journal: British Journal of Cancer (2017)
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
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
Serofendic acid, a neuroprotective substance derived from fetal calf serum, inhibits mitochondrial membrane depolarization and caspase-3 activation
Authors: Kume T, Taguchi R, Katsuki H, Akao M, Sugimoto H, Kaneko S, Akaike A.
Journal: Eur J Pharmacol (2006): 69
Authors: Kume T, Taguchi R, Katsuki H, Akao M, Sugimoto H, Kaneko S, Akaike A.
Journal: Eur J Pharmacol (2006): 69
Multiparameter measurement of caspase 3 activation and apoptotic cell death in NT2 neuronal precursor cells using high-content analysis
Authors: Fennell M, Chan H, Wood A.
Journal: J Biomol Screen (2006): 296
Authors: Fennell M, Chan H, Wood A.
Journal: J Biomol Screen (2006): 296
Asymmetric dimethylarginine induces apoptosis via p38 MAPK/caspase-3-dependent signaling pathway in endothelial cells
Authors: Jiang DJ, Jia SJ, Dai Z, Li YJ.
Journal: J Mol Cell Cardiol (2006): 529
Authors: Jiang DJ, Jia SJ, Dai Z, Li YJ.
Journal: J Mol Cell Cardiol (2006): 529
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
Authors: Xiao XL, Peng J, Su Q, Xiang SL, Tang GH, Huang YS, Zhou XT.
Journal: Ai Zheng (2006): 1247