Green Fluorescent Poly (active) Caspase FAM FLICA® Assay Kit (FAM-VAD-FMK)

  ICT-92 100 Tests

Data Sheet:     SDS:    

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Cell culture  

2-8°C  

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Description

 

This FLICA® (Fluorescent Labeled Inhibitors of CAspases) kit is designed to detect active caspase enzymes. This in vitro assay employs the fluorescent inhibitor probe FAM-VAD-FMK to label active caspase enzymes in living cells. Samples can be analyzed using fluorescence microscopy, a fluorescence plate reader, or by flow cytometry. Our Green Fluorescent Poly (active) Caspase FAM FLICA® Inhibitor Reagent (FAM-VAD-FMK) can be used as a general reagent to detect apoptosis as it is recognized by many types of activated caspases. The probe 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. - 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. - 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.

 

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