AAT Bioquest


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Physical properties
Molecular weight975.04
Spectral properties
Correction Factor (280 nm)0.179
Extinction coefficient (cm -1 M -1)750001
Excitation (nm)554
Emission (nm)578
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure


See also: Caspases
Molecular weight
Correction Factor (280 nm)
Extinction coefficient (cm -1 M -1)
Excitation (nm)
Emission (nm)
TF3-DEVD-FMK is a superior replacement for 5-TAMRA-DEVD-FMK, i.e., 5-carboxytetramethylrhdamine-Asp-Glu-Val-Asp-fluoromethylketone since it can be better excited at 555 nm. TF3 dyes has identical spectral properties to Cy3 with significantly enhanced photostability. TF3-DEVD-FMK binds irreversibly to active caspase 3/7 in stimulated cells. The fluorescent intensity of the TF3-DEVD-FMK signal is proportional to the amount of active caspase 3/7 and can be easily detected by fluorescence microscopy, flow cytometer, or fluorescent microplate reader.

Example protocol


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. 


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.


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of TF3-DEVD-FMK 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 mM102.56 µL512.799 µL1.026 mL5.128 mL10.256 mL
5 mM20.512 µL102.56 µL205.12 µL1.026 mL2.051 mL
10 mM10.256 µL51.28 µL102.56 µL512.799 µL1.026 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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Spectral properties

Correction Factor (280 nm)0.179
Extinction coefficient (cm -1 M -1)750001
Excitation (nm)554
Emission (nm)578



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IL-21R-STAT3 signalling initiates a differentiation program in uterine tissue-resident NK cells to support pregnancy
Authors: Han, Mengwei and Hu, Luni and Wu, Di and Zhang, Yime and Li, Peng and Zhao, Xingyu and Zeng, Yanyu and Ren, Guanqun and Hou, Zhiyuan and Pang, Yanli and others,
Journal: Nature Communications (2023): 7109
The Dietary Flavonoid Fisetin Causes Cell Cycle Arrest, Caspase-Dependent Apoptosis, and Enhanced Cytotoxicity of Chemotherapeutic Drugs in Triple-Negative Breast Cancer Cells
Authors: Smith, M. L., Murphy, K., Doucette, C. D., Greenshields, A. L., Hoskin, D. W.
Journal: J Cell Biochem (2016): 1913-25
Contribution of membrane progesterone receptor alpha to the induction of progesterone-mediated apoptosis associated with mitochondrial membrane disruption and caspase cascade activation in Jurkat cell lines
Authors: Kon, A., Yuan, B., Hanazawa, T., Kikuchi, H., Sato, M., Furutani, R., Takagi, N., Toyoda, H.
Journal: Oncol Rep (2013): 1965-70
Pan-caspase inhibition suppresses polyethylene particle-induced osteolysis
Authors: L, undefined and graeber, S., Jaeckel, S., Loer, F., Wedemeyer, C., Hilken, G., Canbay, A., Totsch, M., von Knoch, M.
Journal: Apoptosis (2009): 173-81
Inhibition of Caspase-Like Activities Prevents the Appearance of Reactive Oxygen Species and Dark-Induced Apoptosis in the Unicellular Chlorophyte Dunaliella Tertiolecta(1)
Authors: Segovia, M., Berges, J. A.
Journal: J Phycol (2009): 1116-26
TNF induces caspase-dependent inflammation in renal endothelial cells through a Rho- and myosin light chain kinase-dependent mechanism
Authors: Wu, X., Guo, R., Chen, P., Wang, Q., Cunningham, P. N.
Journal: Am J Physiol Renal Physiol (2009): F316-26
Leptin induces interleukin-1beta release from rat microglial cells through a caspase 1 independent mechanism
Authors: Pinteaux, E., Inoue, W., Schmidt, L., Molina-Holgado, F., Rothwell, N. J., Luheshi, G. N.
Journal: J Neurochem (2007): 826-33
Silvestrol, a potential anticancer rocaglate derivative from Aglaia foveolata, induces apoptosis in LNCaP cells through the mitochondrial/apoptosome pathway without activation of executioner caspase-3 or -7
Authors: Kim, S., Hwang, B. Y., Su, B. N., Chai, H., Mi, Q., Kinghorn, A. D., Wild, R., Swanson, S. M.
Journal: Anticancer Res (2007): 2175-83
Lipopolysaccharide accelerates caspase-independent but cathepsin B-dependent death of human lung epithelial cells
Authors: Tang, P. S., Tsang, M. E., Lodyga, M., Bai, X. H., Miller, A., Han, B., Liu, M.
Journal: J Cell Physiol (2006): 457-67
C-terminal region of Bfl-1 induces cell death that accompanies caspase activation when fused with GFP
Authors: Yang, W. S., Ko, J. K., Park, S. O., Choi, H. Y., Kim, Y. N., Kim, C. W.
Journal: J Cell Biochem (2005): 1234-47


View all 26 references: Citation Explorer
Suppression of human T cell proliferation by the caspase inhibitors, z-VAD-FMK and z-IETD-FMK is independent of their caspase inhibition properties
Authors: Lawrence CP, Chow SC.
Journal: Toxicol Appl Pharmacol. (2012)
Inhibition of elicitation of allergic contact dermatitis by topical use of Z-VAD-FMK, a broad caspase inhibitor: experiment in mice
Authors: Li YY, Yan CL.
Journal: Zhonghua Yi Xue Za Zhi (2012): 1992
Structure of human caspase-6 in complex with Z-VAD-FMK: New peptide binding mode observed for the non-canonical caspase conformation
Authors: Muller I, Lamers MB, Ritchie AJ, Dominguez C, Munoz-Sanjuan I, Kiselyov A.
Journal: Bioorg Med Chem Lett (2011): 5244
Intracochlear perfusion of leupeptin and z-VAD-FMK: influence of antiapoptotic agents on gunshot-induced hearing loss
Authors: Abaamrane L, Raffin F, Schmerber S, Sendowski I.
Journal: Eur Arch Otorhinolaryngol (2011): 987
Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death
Authors: Meslin B, Beavogui AH, Fasel N, Picot S.
Journal: PLoS One (2011): e23867
Pretreatment with pancaspase inhibitor (Z-VAD-FMK) delays but does not prevent intraperitoneal heat-killed group B Streptococcus-induced preterm delivery in a pregnant mouse model
Authors: Equils O, Moffatt-Blue C, Ishikawa TO, Simmons CF, Ilievski V, Hirsch E.
Journal: Infect Dis Obstet Gynecol (2009): 749432
Attenuation of allergic contact dermatitis by Z-VAD-FMK, a broad caspase inhibitor: experiment with mice
Authors: Li YY, Yan CL, Xu W.
Journal: Zhonghua Yi Xue Za Zhi (2008): 3153
Caspase inhibitor Z-VAD-FMK enhances the freeze-thaw survival rate of human embryonic stem cells
Authors: Heng BC, Clement MV, Cao T.
Journal: Biosci Rep (2007): 257
Effects of caspase inhibitors (z-VAD-fmk, z-VDVAD-fmk) on Nile Red fluorescence pattern in 7-ketocholesterol-treated cells: investigation by flow cytometry and spectral imaging microscopy
Authors: Vejux A, Lizard G, Tourneur Y, Riedinger JM, Frouin F, Kahn E.
Journal: Cytometry A (2007): 550
Bodipy-VAD-Fmk, a useful tool to study yeast peptide N-glycanase activity
Authors: Witte MD, Descals CV, de Lavoir SV, Florea BI, van der Marel GA, Overkleeft HS.
Journal: Org Biomol Chem (2007): 3690