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Chemical structure for FITC-C6-LEHD-FMK
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Ex/Em (nm)492/516
Storage Freeze (<-15 °C)
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Category Enzyme Detection
Peptidases and Proteases
Related Cell Apoptosis
Apoptosis and Cytotoxicity
Secondary Reagents
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.

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.

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Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
/ = x =

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This protocol only provides a guideline, and should be modified according to your specific needs.

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

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

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

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

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

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

Protein kinase Cdelta amplifies ceramide formation via mitochondrial signaling in prostate cancer cells
Authors: Sumitomo M, Ohba M, Asakuma J, Asano T, Kuroki T, Hayakawa M.
Journal: J Clin Invest (2002): 827

3-m-bromoacetylamino benzoic acid ethyl ester: a new cancericidal agent that activates the apoptotic pathway through caspase-9
Authors: Schlesinger M, Jiang JD, Roboz JP, Denner L, Ling YH, Holland JF, Bekesi JG.
Journal: Biochem Pharmacol (2000): 1693

Human malignant glioma therapy using anti-alpha(v)beta3 integrin agents
Authors: Chatterjee S, Matsumura A, Schradermeier J, Gillespie GY.
Journal: J Neurooncol (2000): 135

Phenylephrine protects neonatal rat cardiomyocytes from hypoxia and serum deprivation-induced apoptosis
Authors: Zhu H, McElwee-Witmer S, Perrone M, Clark KL, Zilberstein A.
Journal: Cell Death Differ (2000): 773

Additional Documents

Safety Data Sheet (SDS)

1. Enzyme Probes & Assay Kits

Certificate of Analysis