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Fluorometric detection of active caspases 3/7 using FITC-C6-D(OMe)E(OMe)VD(OMe)-FMK (Cat# 13418) in Jurkat cells. The cells were treated with 1 μM staurosporine for 4 hours (Green) while untreated cells were used as a control (Red). Control or treated Cells were incubated with FITC-C6-D(OMe)E(OMe)VD(OMe)-FMK for 1 hour at 37 °C, and then washed once after stain.  Fluorescent intensity was measured with NovoCyte™ 3000 Flow Cytometer FITC channel.
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13417 $195

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Ex/Em (nm)492/516
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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-D(OMe)E(OMe)VD(OMe)-FMK provides a convenient means for sensitive detection of activated caspase-3 in living cells. FITC-C6-D(OMe)E(OMe)VD(OMe)-FMK is cell permeable, nontoxic, and irreversibly binds to activated caspase-3 in apoptotic cells. Based on our in-house studies FITC-C6-D(OMe)E(OMe)VD(OMe)-FMK gave the highest signal/background ratio compared to other DEVD-FMK probes we tested. 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-D(OMe)E(OMe)VD(OMe)-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

Activation of caspase-like activity and poly (ADP-ribose) polymerase degradation during sporulation in Aspergillus nidulans
Authors: Thrane C, Kaufmann U, Stummann BM, Olsson S.
Journal: Fungal Genet Biol (2004): 361

[Antitumor mechanism of 3-bromopropionylamino benzoylurea on leukemia and lymphoma]
Authors: Li JN, Song DQ, Jiang JD.
Journal: Yao Xue Xue Bao (2004): 491

Caspase-dependent and independent cell death in rat hepatoma 5123tc cells
Authors: Pandey S, Smith B, Walker PR, Sikorska M.
Journal: Apoptosis (2000): 265

C-myc antisense oligodeoxynucleotides can induce apoptosis and down-regulate Fas expression in rheumatoid synoviocytes
Authors: Hashiramoto A, Sano H, Maekawa T, Kawahito Y, Kimura S, Kusaka Y, Wilder RL, Kato H, Kondo M, Nakajima H.
Journal: Arthritis Rheum (1999): 954

Additional Documents

Safety Data Sheet (SDS)

1. Enzyme Probes & Assay Kits

Certificate of Analysis