Amplite® Fluorimetric Caspase 3/7 Assay Kit *Blue Fluorescence*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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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 | ![]() ![]() |
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
![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).](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-fluorimetric-caspase-3-7-assay-kit-blue-fluorescence%2Ffigure-for-amplite-fluorimetric-caspase-3-7-assay-kit-blue-fluorescence_gHUqg.jpg&w=3840&q=75)
![<strong>Effect of TA chimeras on liposomal Dox-mediated activation of caspase 3/7. </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 < 0.05). Source: <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.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-fluorimetric-caspase-3-7-assay-kit-blue-fluorescence%2Ffigure-for-amplite-fluorimetric-caspase-3-7-assay-kit-blue-fluorescence_gzhHJ.jpg&w=3840&q=75)
![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 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 cm) were evaluated. STAT3 activation (D, scale bar: 20 μm), caspase-3 activation (E, scale bar: 200 μm), 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 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>, July 2016.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-fluorimetric-caspase-3-7-assay-kit-blue-fluorescence%2Ffigure-for-amplite-fluorimetric-caspase-3-7-assay-kit-blue-fluorescence_6JrcR.jpg&w=3840&q=75)
![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 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 hours, followed by caspase-3 measurement (B). The data are presented as the mean ± SD. *p-value < 0.05, **p-value < 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 hours, followed by the measurement of pSTAT3, STAT3 and β-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>, July 2016.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Famplite-fluorimetric-caspase-3-7-assay-kit-blue-fluorescence%2Ffigure-for-amplite-fluorimetric-caspase-3-7-assay-kit-blue-fluorescence_i6S4Y.jpg&w=3840&q=75)
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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,
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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: 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
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