Green Fluorescent FAM-FLICA® Caspase-9 (LEHD) Assay Kit

This in vitro assay employs the green fluorescent inhibitor probe FAM-LEHD-FMK to label active caspase-9 enzyme in living cells. Analyze samples using fluorescence microscopy, a fluorescence plate reader, or flow cytometry.

SKU: 912

Size: 25 Tests
Sale price$216.00

Our FLICA® probes are non-cytotoxic Fluorescent Labeled Inhibitors of CAspases that covalently bind to active caspase enzymes. FLICA® measures the intracellular process of apoptosis instead of a side-effect, such as the turnover of phosphatidyl serine, and eliminates the incidence of false positives that often plagues methods like Annexin V and TUNEL staining. FLICA® can also be used to measure pyroptosis, a highly inflammatory form of programmed cell death.

To use FLICA®, add it directly to the cell culture media, incubate, and wash. FLICA® is cell-permeant and will efficiently diffuse in and out of all cells. If there is an active caspase enzyme inside the cell, it will covalently bind with FLICA® and retain the green fluorescent signal within the cell. Unbound FLICA® will diffuse out of the cell during the wash steps. Apoptotic and pyroptotic cells will retain a higher concentration of FLICA® and fluoresce brighter than healthy cells. There is no interference from pro-caspases or inactive forms of the enzymes. If the test treatment is causing cell death via apoptosis and/or pyroptosis, the cells will contain an elevated level of caspase activity relative to negative control cells and fluoresce with FLICA®.

Apoptosis is an evolutionarily conserved process of programmed cell suicide. It is centered on a cascade of proteolytic enzymes called caspases that are triggered in response to pro-apoptotic signals. Once activated, caspases cleave protein substrates leading to the eventual disassembly of the cell. Caspases have been identified in organisms ranging from C. elegans to humans. Mammalian caspases play distinct roles in both apoptosis and inflammation. In apoptosis, effector caspases (-3, -6, and -7) are responsible for proteolytic cleavages that lead to cell disassembly. Initiator caspases (-8, -9, and -10) regulate apoptosis upstream. Caspase-1 is associated with pyroptosis and inflammasome activity and takes on the role of a key housekeeping enzyme in its conversion of pro-IL-1ß protein into the active IL-1ß cytokine (Use FLICA® kits #98, #9122, and #9162 to detect caspase-1). Please note that macrophages and monocytes have been shown to rapidly secrete caspase-1 upon activation.

Like the majority of other proteases, caspases are synthesized as pro-form precursors that undergo proteolytic maturation, either autocatalytically or in a cascade by enzymes with similar specificity. Active caspase enzymes consist of two large (~20 kD) and two small (~10 kD) subunits that non-covalently associate to form a two heterodimer, tetrameric active caspase. Activated caspase enzymes cleave proteins by recognizing a 3 or 4 amino acid sequence that must include an aspartic acid (D) residue in the P1 position. This C–terminal residue is the target for the cleavage reaction at the carbonyl end. Each FLICA® probe contains a 3 or 4 amino acid sequence that is targeted by different activated caspases. This target sequence is sandwiched between a green fluorescent label, carboxyfluorescein (FAM), and a fluoromethyl ketone (FMK). Caspases cannot cleave the FLICA® inhibitor probe; instead, they form an irreversible covalent bond with the FMK on the target sequence and enzyme activity is inhibited. Our poly caspase FLICA® probe, FAM-VAD-FMK, can be used as a general reagent to detect apoptosis as it is recognized by many types of activated caspases. To more specifically target a particular caspase enzyme, use one of our specialized FLICA® reagents. We have kits for the detection of: caspase-1 (YVAD or WEHD) (also recognizes caspases 4 and 5), -2 (VDVAD), -3/7 (DEVD), -6 (VEID), -8 (LETD), -9 (LEHD), and -10 (AEVD). FLICA® kits are also available with a red or far red fluorescent label. Caspases, like most other crucial cell survival enzymes, are somewhat permissive in the target amino acid sequence they will recognize and cleave. Therefore, although FLICA® reagents contain the different amino acid target sequences preferred by each caspase, they can also recognize other active caspases when they are present. We encourage validation of caspase activity by an orthogonal technique.

FLICA® can be used to label suspension or adherent cells and thin tissue sections. After labeling with FAM-FLICA®, cells can be fixed or frozen. For tissues that will be paraffin-embedded after labeling, use our red sulforhodamine SR-FLICA® probes; do not use the green FAM-FLICA® probes as the FAM dye will be quenched during the paraffin embedding process.

Cells labeled with FAM-FLICA® can be counter-stained with reagents such as the red live/dead stains Propidium Iodide (included in FAM-FLICA® kits) and 7-AAD (catalog # 6163) to distinguish apoptosis from necrosis. Nuclear morphology can be concurrently observed using Hoechst 33342, a blue DNA binding dye (included in FLICA® kits). Cells can be viewed directly through a fluorescence microscope, or the fluorescence intensity can be quantified using a flow cytometer or fluorescence plate reader. FAM-FLICA® optimally excites at 488-492 nm and has a peak emission at 515-535 nm.

488 nm / 530 nm
Flow Cytometry, Fluorescence Microscope, Fluorescence Plate Reader
Cell culture
Domestic: Overnight Delivery; International: Priority Shipping
United States
  1. Prepare samples and controls
  2. Dilute 10X Apoptosis Wash Buffer 1:10 with diH20.
  3. Reconstitute FLICA with 50 μL DMSO.
  4. Dilute FLICA 1:5 by adding 200 μL PBS.
  5. Add diluted FLICA to each sample at 1:30 (e.g., add 10 μL to 290 μL of cultured cells).
  6. Incubate approximately 1 hour.
  7. Remove media and wash cells 3 times: add 1X Apoptosis Wash Buffer and spin cells.
  8. If desired, label with additional stains, such as Hoechst, Propidium Iodide, 7-AAD, or an antibody.
  9. If desired, fix cells.
  10. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. FAM FLICA excites at 492 nm and emits at 520 nm.

If working with adherent cells, please see the manual for additional protocols.

Kit 912: 25 Tests
  • FLICA Caspase-9 Reagent (FAM-LEHD-FMK), 1 vial, #677
  • 10X Apoptosis Wash Buffer, 15 mL, #635
  • Fixative, 6 mL, #636
  • Propidium Iodide, 1 mL, #638
  • Hoechst 33342, 1 mL, #639
  • Kit Manual
  • Kit 913: 100 tests
  • FLICA Caspase-9 Reagent (FAM-LEHD-FMK), 4 vials, #677
  • 10X Apoptosis Wash Buffer, 60 mL, #634
  • Fixative, 6 mL, #636
  • Propidium Iodide, 1 mL, #638
  • Hoechst 33342, 1 mL, #639
  • Kit Manual
  • Product Specific References

    PMID Publication
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    36768363Krętowski, R., et al. 2023. The Effect of Silica Nanoparticles (SiNPs) on Cytotoxicity, Induction of Oxidative Stress and Apoptosis in Breast Cancer Cell Lines. International Journal of Molecular Sciences, 2037.
    37001390Ivasechko, I., et al. 2023. Molecular design, synthesis and anticancer activity of new thiopyrano[2,3-d]thiazoles based on 5-hydroxy-1,4-naphthoquinone (juglone). European journal of medicinal chemistry, 115304.
    37763082Mańka, S., et al. 2023. Cytotoxic Activity of Melatonin Alone and in Combination with Doxorubicin and/or Dexamethasone on Diffuse Large B-Cell Lymphoma Cells in In Vitro Conditions. Journal of Personalized Medicine, 1314.
    35055021Rok, J., et al. 2022. The Anticancer Potential of Doxycycline and Minocycline-A Comparative Study on Amelanotic Melanoma Cell Lines. International journal of molecular sciences.
    34994998Chiu, Y.J., et al. 2022. Curcumin suppresses cell proliferation and triggers apoptosis in vemurafenib-resistant melanoma cells by downregulating the EGFR signaling pathway. Environmental toxicology.
    35270004González-Sarrías, A., et al. 2022. Milk-Derived Exosomes as Nanocarriers to Deliver Curcumin and Resveratrol in Breast Tissue and Enhance Their Anticancer Activity. International journal of molecular sciences.
    35640691Ophélie, G., et al. 2022. Cellular and molecular mechanisms of NiONPs toxicity on eel hepatocytes HEPA-E1: An illustration of the impact of Ni release from mining activity in New Caledonia. Chemosphere, 135158.
    35467089Kubiak, A.B., et al. 2022. The influence of venetoclax, used alone or in combination with cladribine (2-CdA), on CLL cells apoptosis in vitro: Preliminary results. Advances in clinical and experimental medicine : official organ Wroclaw Medical University.
    35842415Trugilho, M.R.O., et al. 2022. Platelet proteome reveals features of cell death, antiviral response and viral replication in covid-21. Cell death discovery, 324.
    36012549Krętowski, R., et al. 2022. The Reduced Graphene Oxide (rGO) Induces Apoptosis, Autophagy and Cell Cycle Arrest in Breast Cancer Cells. International journal of molecular sciences.

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