Amplite® Fluorimetric Caspase 3/7 Assay Kit *Red Fluorescence*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Excitation (nm) | 532 |
Emission (nm) | 619 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12352200 |
Overview | SDSProtocol |
Excitation (nm) 532 | Emission (nm) 619 |
Platform
Fluorescence microplate reader
Excitation | 535 nm |
Emission | 620 nm |
Cutoff | 610 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells with test compounds
- Add equal volume of caspase 3/7 working solution
- Incubate at room temperature for 1 hour
- Monitor fluorescence intensity at Ex/Em = 535/620 nm
Important notes
Thaw Component A, B, C (if desired, Component D) at room temperature before use.
PREPARATION OF STOCK SOLUTION
1. Z-DEVD-ProRed™ stock solution (200X):
Add 65 µL of DMSO (not provided) into the vial of Component A.
2. (Optional) Caspase 3/7 Inhibitor Ac-DEVD-CHO stock solution (1 mM):
Add 100 µL of DMSO directly to the vial of 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 Z-DEVD-ProRed™ stock solution and 100 μL of 1M DTT solution (Component C) into 10 mL Assay Buffer (Component B) and mix well.
Note: 50 μL of the 200X Z-DEVD-ProRed™ stock solution is enough for 100 assays using a reaction volume of 100 μL per assay.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
- Treat cells by adding 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-plate) into PBS or desired buffer. For blank wells (medium without the cells), add the same amount of compound buffer.
- Incubate the cell plates in an incubator for a desired period of time (3 - 5 hours for Jurkat cells treated with staurosporine) to induce apoptosis.
- 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, kept from light. Note: If desired, add 1 µL of the 1 mM stock solution of the caspase 3/7 Inhibitor Ac-DEVD-CHO into selected samples 10 minutes before adding the caspase 3/7 assay working solution at room temperature to confirm the caspase 3/7-like activities.
- Monitor the fluorescence intensity at Ex/Em = 535/620 nm (cut off at 610 nm) with either top or bottom read mode. Note: Sometimes, bottom read gives better signal to background ratio, centrifuge cell plate (especially for the nonadherent cells) at 800 rpm for 2 minutes (brake off) if using bottom read mode.
Product Family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) |
Amplite® Fluorimetric Caspase 3/7 Assay Kit *Blue Fluorescence* | 341 | 441 | - |
Amplite® Fluorimetric Caspase 3/7 Assay Kit *Green Fluorescence* | 500 | 522 | 80000 |
Images
Citations
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
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)
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
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
Authors: Onodera, Risako and Morioka, Shunsuke and Unida, Shinshu and Motoyama, Keiichi and Tahara, Kohei and Takeuchi, Hirofumi
Journal: European Journal of Pharmaceutical Sciences (2022): 106081
Authors: Suzuki, Ryusuke and Fujiwara, Yukio and Saito, Mitsuru and Arakawa, Shoutaro and Shirakawa, Jun-ichi and Yamanaka, Mikihiro and Komohara, Yoshihiro and Marumo, Keishi and Nagai, Ryoji
Journal: Journal of bone and mineral research (2020): 1992--2003
Authors: Tavares, Raquel and Pathak, Sushil Kumar
Journal: Frontiers in Cellular and Infection Microbiology (2017): 58
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: Jiang, Jiaojiao and Hu, Jianzhong and Xie, Zeyi and Cao, Qinghe and Ma, Daifu and Han, Yonghua and Li, Zongyun
Journal: Journal of Rare Earths (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,
Journal: British journal of cancer (2017): 1177--1185
References
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.
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Journal: Biochem Biophys Res Commun (2006): 878
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Journal: Neurotoxicology (2006): 44
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Journal: J Biomol Screen (2006): 296
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Journal: Eur J Pharmacol (2006): 69
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