Amplite® Fluorimetric Caspase 3/7 Assay Kit *Blue Fluorescence*
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Unit Size | |
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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) | 341 |
Emission (nm) | 441 |
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) 341 | Emission (nm) 441 |
Platform
Fluorescence microplate reader
Excitation | 350 nm |
Emission | 450 nm |
Cutoff | 420 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells with test compounds (100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate)
- Add equal volume of Caspase 3/7 assay working solution
- Incubate at room temperature for 1 hour
- 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
(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
- 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.
- 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.
- 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. 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.
- Centrifuge the cell plate (especially for the non-adherent cells) at 800 rpm for 2 minutes with brake off.
- Monitor the fluorescence increase at Ex/Em = 350/450 nm.
Product Family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) |
Amplite® Fluorimetric Caspase 3/7 Assay Kit *Green Fluorescence* | 500 | 522 | 80000 |
Amplite® Fluorimetric Caspase 3/7 Assay Kit *Red Fluorescence* | 532 | 619 | - |
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: 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
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: Elaskalani, Omar and Domenchini, Alice and Abdol Razak, Norbaini Binti and E Dye, Danielle and Falasca, Marco and Metharom, Pat
Journal: Cancers (2020): 250
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: Abdelrehim, Abbi and Shaltiel, Lior and Zhang, Ling and Barenholz, Yechezkel and High, Stephen and Harris, Lynda K
Journal: PloS one (2019): e0212701
Authors: Yamamoto, Takeshi and Kida, Yutaka and Kuwano, Koichi
Journal: Cellular microbiology (2019): e13015
References
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Journal: J Biomol Screen (2006): 296
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Journal: Eur J Pharmacol (2006): 69
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