FITC-C6-LEHD-FMK
Activation of caspases plays a central role in apoptosis. FITC-C6-LEHD-FMK provides a convenient means for sensitive detection of activated caspase-9 in living cells. FITC-LEHD-FMK is cell permeable, nontoxic, and irreversibly binds to activated caspase-9 in apoptotic cells. The FITC label allows for direct detection of activated caspases in apoptotic cells by fluorescence microscopy, flow cytometry, or fluorescence plate reader.
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 FITC-C6-LEHD-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 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 96.987 µL | 484.933 µL | 969.866 µL | 4.849 mL | 9.699 mL |
5 mM | 19.397 µL | 96.987 µL | 193.973 µL | 969.866 µL | 1.94 mL |
10 mM | 9.699 µL | 48.493 µL | 96.987 µL | 484.933 µL | 969.866 µL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
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Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield | Correction Factor (280 nm) |
FITC-C6-DEVD-FMK | 491 | 516 | 73000 | 0.92 | 0.35 |
References
View all 9 references: Citation Explorer
Endoplasmic reticulum stress and trophic factor withdrawal activate distinct signaling cascades that induce glycogen synthase kinase-3 beta and a caspase-9-dependent apoptosis in cerebellar granule neurons
Authors: Brewster JL, Linseman DA, Bouchard RJ, Loucks FA, Precht TA, Esch EA, Heidenreich KA.
Journal: Mol Cell Neurosci (2006): 242
Authors: Brewster JL, Linseman DA, Bouchard RJ, Loucks FA, Precht TA, Esch EA, Heidenreich KA.
Journal: Mol Cell Neurosci (2006): 242
Enhanced 15-HPETE production during oxidant stress induces apoptosis of endothelial cells
Authors: Sordillo LM, Weaver JA, Cao YZ, Corl C, Sylte MJ, Mullarky IK.
Journal: Prostaglandins Other Lipid Mediat (2005): 19
Authors: Sordillo LM, Weaver JA, Cao YZ, Corl C, Sylte MJ, Mullarky IK.
Journal: Prostaglandins Other Lipid Mediat (2005): 19
P2X7 receptor-mediated apoptosis of human cervical epithelial cells
Authors: Wang Q, Wang L, Feng YH, Li X, Zeng R, Gorodeski GI.
Journal: Am J Physiol Cell Physiol (2004): C1349
Authors: Wang Q, Wang L, Feng YH, Li X, Zeng R, Gorodeski GI.
Journal: Am J Physiol Cell Physiol (2004): C1349
Induction of apoptosis by apicidin, a histone deacetylase inhibitor, via the activation of mitochondria-dependent caspase cascades in human Bcr-Abl-positive leukemia cells
Authors: Cheong JW, Chong SY, Kim JY, Eom JI, Jeung HK, Maeng HY, Lee ST, Min YH.
Journal: Clin Cancer Res (2003): 5018
Authors: Cheong JW, Chong SY, Kim JY, Eom JI, Jeung HK, Maeng HY, Lee ST, Min YH.
Journal: Clin Cancer Res (2003): 5018
Caspase activation during Haemophilus somnus lipooligosaccharide-mediated apoptosis of bovine endothelial cells
Authors: Sylte MJ, Leite FP, Kuckleburg CJ, Inzana TJ, Czuprynski CJ.
Journal: Microb Pathog (2003): 285
Authors: Sylte MJ, Leite FP, Kuckleburg CJ, Inzana TJ, Czuprynski CJ.
Journal: Microb Pathog (2003): 285
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