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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

  1. Prepare a 10 mM stock solution in DMSO.

  2. 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.

  3. 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.

  4. Monitor the fluorescence using a fluorescence microplate reader, or absorbance using an absorbance microplate reader.

Cell Caspase Assays Using Cell-Permeable FMK Caspase Probes

  1. Prepare a 2-5 mM stock solution in DMSO.

  2. Treat cells as desired.

  3. 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).

  4. 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.

  5. Wash the cells with HHBS for at least once.

  6. 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)

  1. Prepare a 250X stock solution by adding 50 µL DMSO into the vial.

  2. Treat cells as desired.

  3. 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.

  4. Wash the cells with HHBS for at least once.

  5. 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 mg0.5 mg1 mg5 mg10 mg
1 mM137.06 µL685.298 µL1.371 mL6.853 mL13.706 mL
5 mM27.412 µL137.06 µL274.119 µL1.371 mL2.741 mL
10 mM13.706 µL68.53 µL137.06 µL685.298 µL1.371 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum

Product family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)
Ac-DEVD-AMC *CAS 169332-61-0*341441-
Ac-IETD-AFC *CAS 211990-57-7*376482170001
Z-DEVD-AFC376482170001
Ac-DEVD-aminoluciferin36249918000

Citations

View all 7 citations: Citation Explorer
Modeling ferroptosis in human dopaminergic neurons: pitfalls and opportunities for neurodegeneration research
Authors: Renner, Nadine and Sch{\"o}b, Franziska and Pape, Regina and Suciu, Ilinca and Spreng, Anna-Sophie and {\"U}ckert, Anna-Katharina and C{\"o}llen, Eike and Bovio, Federica and Chilian, Bruno and Bauer, Johannes and others,
Journal: Redox Biology (2024): 103165
Eriobotrya japonica Fermentation with Plant-Derived Lactiplantibacillus plantarum MSC-5T Ameliorates Antioxidant Activity in HEK293 Cells
Authors: Danshiitsoodol, Narandalai and Inoue, Yusuke and Sugimoto, Sachiko and Shakya, Shrijana and Noda, Masafumi and Sugiyama, Masanori
Journal: Fermentation (2024): 197
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

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
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
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
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
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
Page updated on November 3, 2024

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Physical properties

Molecular weight

729.61

Solvent

DMSO

Spectral properties

Extinction coefficient (cm -1 M -1)

170001

Excitation (nm)

376

Emission (nm)

482

Quantum yield

0.531

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

CAS

201608-14-2
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