Ac-DEVD-AFC *CAS 201608-14-2*
Ac-DEVD-AFC is a fluorogenic substrate for caspase 3, a protease that is rapidly activated when cells are exposed to apoptotic conditions and that cleaves poly(ADP-ribose) polymerase. The 7-amido-4-trifluoromethylcoumarin derivatives have better membrane permeability than the 7-amido-4-methylcoumarin derivatives.
Example protocol
AT A GLANCE
Important notes
It is important to store at <-15 °C and should be stored in cool, dark place.
It can be used within 12 months from the date of receipt.
SAMPLE EXPERIMENTAL PROTOCOL
Following protocol only provides a guideline, and should be modified according to your specific needs.
General Solution Caspase Assays Using AMC, AFC, pNA, R110 and ProRed Substrates
- Prepare a 10 mM stock solution in DMSO.
- Prepare a 2X caspase substrate (50 µM) assay solution as the following: 50 µL substrate stock solution, 100 µL DTT (1M), 400 µL EDTA (100 mM), 10 mL Tris Buffer (20 mM), pH =7.4.
- Mix equal volume of the caspase standards or samples with 2X caspase substrate assay solution, and incubate the solutions at room temperature for at least 1 hour.
- Monitor the fluorescence using a fluorescence microplate reader, or absorbance using an absorbance microplate reader.
Cell Caspase Assays Using Cell-Permeable FMK Caspase Probes
- Prepare a 2-5 mM stock solution in DMSO.
- Treat cells as desired.
- Prepare a 2X permeable caspase substrate (20 µM) assay solution by diluting the DMSO stock solution (from Step 2.1) in Hanks with 20 mM Hepes buffer (HHBS).
- Mix equal volume of the treated cells with 2X caspase substrate assay solution (from Step 2.3), and incubate the cells in a 37°C, 5% CO2 incubator for at least1 hour.
- Wash the cells with HHBS for at least once.
- Monitor the fluorescence intensity by a flow cytometer, a fluorescence microscope or a fluorescence microplate reader.
Cell Caspase Assays Using Cell-Permeable FMK Caspase Probes (For #13470-13476 only)
- Prepare a 250X stock solution by adding 50 µL DMSO into the vial.
- Treat cells as desired.
- Add 250 X DMSO stock solution into the cell solution at a 1:250 ratio (such as 2 µL to 500 µL cells), and incubate the cells in a 37°C, 5% CO2 incubator for 1 hour.
- Wash the cells with HHBS for at least once.
- Monitor the fluorescence intensity by flow cytometer, fluorescence microscopy or fluorescent microplate reader.
Calculators
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of Ac-DEVD-AFC *CAS 201608-14-2* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 137.06 µL | 685.298 µL | 1.371 mL | 6.853 mL | 13.706 mL |
5 mM | 27.412 µL | 137.06 µL | 274.119 µL | 1.371 mL | 2.741 mL |
10 mM | 13.706 µL | 68.53 µL | 137.06 µL | 685.298 µL | 1.371 mL |
Molarity calculator
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Spectrum
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Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) |
Ac-DEVD-AMC *CAS 169332-61-0* | 341 | 441 | - |
Ac-IETD-AFC *CAS 211990-57-7* | 376 | 482 | 170001 |
Z-DEVD-AFC | 376 | 482 | 170001 |
Ac-DEVD-aminoluciferin | 362 | 499 | 18000 |
Citations
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Journal: Redox Biology (2024): 103165
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Journal: Redox Biology (2024): 103165
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Mitophagy regulates mitochondrial network signaling, oxidative stress, and apoptosis during myoblast differentiation
Authors: Baechler, Brittany L and Bloemberg, Darin and Quadrilatero, Joe
Journal: Autophagy (2019): 1606--1619
Authors: Baechler, Brittany L and Bloemberg, Darin and Quadrilatero, Joe
Journal: Autophagy (2019): 1606--1619
Regulation of glioblastoma cell proliferation in dependence of cell density and growth factors in vitro
Authors: Liu, Yun
Journal: (2019)
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Journal: (2019)
SOX9 in the development and chemotherapy of cholangiocarcinoma (CCA)
Authors: Yuan, Xiaodong
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References
View all 67 references: Citation Explorer
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
Authors: Sakaue M, Motoyama Y, Yamamoto K, Shiba T, Teshima T, Chiba K.
Journal: Biochem Biophys Res Commun (2006): 878
Serofendic acid, a neuroprotective substance derived from fetal calf serum, inhibits mitochondrial membrane depolarization and caspase-3 activation
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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
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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
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Diallyl Trisulfide Induces Apoptosis of Human Gastric Cancer Cell Line MGC803 Through Caspase-3 Pathway.
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Journal: Ai Zheng (2006): 1247
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