| Overview |
 Printer Friendly Version
|
| Ex/Em (nm) | 492/518 |
| MW | 691.66 |
| CAS # | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category |
Enzyme Detection Peptidases and Proteases |
| Related |
Cell Apoptosis Apoptosis and Cytotoxicity Biochemical Assays |
| Spectrum | Advanced Spectrum Viewer |
1. General Solution Caspase Assays Using AMC, AFC, pNA, R110 and ProRed Substrates
1.1. Prepare a 10 mM stock solution in DMSO.
1.2. 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
1.3. Mix equal volume of the caspase standards or samples with 2X caspase substrate assay solution (from Step 1.2), and incubate the solutions at room temperature for at least 1 hour.
1.4. Monitor the fluorescence using a fluorescence microplate reader, or absorbance using an absorbance microplate reader.
2. Cell Caspase Assays Using Cell-Permeable FMK Caspase Probes
2.1. Prepare a 2-5 mM stock solution in DMSO.
2.2. Treat cells as desired.
2.3. 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).
2.4. 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.
2.5. Wash the cells with HHBS for at least once.
2.6. Monitor the fluorescence intensity by a flow cytometer, a fluorescence microscope or a fluorescence microplate reader.
3. Cell Caspase Assays Using Cell-Permeable FMK Caspase Probes (For #13470-13476 only)
3.1. Prepare a 250X stock solution by adding 50 µL DMSO into the vial.
3.2. Treat cells as desired.
3.3. Add 250 X DMSO stock solution (from Step 3.1) into the cell solution at a 1:250 ratio (such as 2 uL to 500 uL cells), and incubate the cells in a 37°C, 5% CO2 incubator for 1 hour.
3.4. Wash the cells with HHBS for at least once.
3.5. Monitor the fluorescence intensity by flow cytometer, fluorescence microscopy or fluorescent microplate reader.
| References & Citations |
 Printer Friendly Version
|
1. Lawrence CP, Chow SC. (2012) Suppression of human T cell proliferation by the caspase inhibitors, z-VAD-FMK and z-IETD-FMK is independent of their caspase inhibition properties. Toxicol Appl Pharmacol.
2. Li YY, Yan CL. (2012) [Inhibition of elicitation of allergic contact dermatitis by topical use of Z-VAD-FMK, a broad caspase inhibitor: experiment in mice]. Zhonghua Yi Xue Za Zhi, 92, 1992.
3. Meslin B, Beavogui AH, Fasel N, Picot S. (2011) Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death. PLoS One, 6, e23867.
4. Muller I, Lamers MB, Ritchie AJ, Dominguez C, Munoz-Sanjuan I, Kiselyov A. (2011) Structure of human caspase-6 in complex with Z-VAD-FMK: New peptide binding mode observed for the non-canonical caspase conformation. Bioorg Med Chem Lett, 21, 5244.
5. Abaamrane L, Raffin F, Schmerber S, Sendowski I. (2011) Intracochlear perfusion of leupeptin and z-VAD-FMK: influence of antiapoptotic agents on gunshot-induced hearing loss. Eur Arch Otorhinolaryngol, 268, 987.
6. Equils O, Moffatt-Blue C, Ishikawa TO, Simmons CF, Ilievski V, Hirsch E. (2009) 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. Infect Dis Obstet Gynecol, 2009, 749432.
7. Li YY, Yan CL, Xu W. (2008) [Attenuation of allergic contact dermatitis by Z-VAD-FMK, a broad caspase inhibitor: experiment with mice]. Zhonghua Yi Xue Za Zhi, 88, 3153.
8. Witte MD, Descals CV, de Lavoir SV, Florea BI, van der Marel GA, Overkleeft HS. (2007) Bodipy-VAD-Fmk, a useful tool to study yeast peptide N-glycanase activity. Org Biomol Chem, 5, 3690.
9. Heng BC, Clement MV, Cao T. (2007) Caspase inhibitor Z-VAD-FMK enhances the freeze-thaw survival rate of human embryonic stem cells. Biosci Rep, 27, 257.
10. Vejux A, Lizard G, Tourneur Y, Riedinger JM, Frouin F, Kahn E. (2007) 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. Cytometry A, 71, 550.
