Description
Description: Detect active caspase-8 with the green fluorescence FAM-FLICA Caspase 8 Assay Kit. This in vitro apoptosis assay employs the fluorescent inhibitor probe FAM-LETD-FMK to label active caspase-8 enzymes in living cells or tissue samples. Analyze the fluorescent signal using fluorescence microscopy, a fluorescent plate reader, or by flow cytometry. FLICA (Fluorescent Labeled Inhibitor of Caspases) probes are comprised of an inhibitor peptide sequence that binds to active caspase enzymes, a fluoromethyl ketone (FMK) moiety that results in an irreversible binding event with the enzyme, and a fluorescent tag (either carboxyfluorescein or sulforhodamine B) reporter. For a caspase-8 inhibitor, the enzyme recognition sequence is leucine-glutamic acid-threonine-aspartic acid (LETD). The FLICA probe interacts with the enzymatic reactive center of an activated caspase via the recognition sequence, forming a covalent thioether adduct with the enzyme through the FMK moiety. FLICA probes are cell permeant and non-cytotoxic. Unbound FAM-FLICA reagent is washed away; the remaining green fluorescent signal is a direct measure of caspase-8 activity at the time the probe was added. Detection of nuclear morphology and necrosis is also possible with the additional kit components Hoechst 33342 and Propidium Iodide.
Background: poptosis is an evolutionarily conserved form of cell suicide mediated by a cascade of proteolytic enzymes called caspases, or cysteinyl aspartate-specific proteases. Pro-apoptotic signals activate the enzymatic cascade resulting in the cleavage of protein substrates, leading to the disassembly of the cell (1-4). Caspases have been identified in organisms ranging from C. elegans to humans. Members of the mammalian caspase family of cysteinyl aspartate- specific proteases play distinct roles in apoptosis and inflammation. Caspases are categorized in two groups: the initiators (caspases 8, 9, and 10) and the effector caspases (caspases 1, 2, 3, 4, 6, 7, 12, and 13). The initiator caspases 8 and 10 are involved in what is referred to as the extrinsic apoptosis pathway that originates upon activation of cell surface death receptors. These monomers bind to death receptor proteins through their death effector domain (DED) structure. Initiator caspase 9 is involved in the intrinsic apoptosis pathway that results from the mitochondrial release of cytochrome c. The caspase 9 monomer binds other proteins through their caspase activation and recruitment domain (CARD). These initiator caspase-protein interactions result in dimerization of the initiator caspases that leads to their activation. The activated initiator caspases then cleave the effector pro-caspases at specific aspartic acid residues to yield large (20 kDa) and small (10 kDa) subunits that then assemble into the heterotetrameric, catalytically active forms of the effector caspase enzymes (5, 6). Active caspase enzymes exhibit catalytic and substrate specificities comprised of short tetra-peptide amino acid sequences that must contain an aspartate in the P1 position (7 - 9). These preferred tetra-peptide sequences have been used to derive peptides that specifically compete for caspase binding (4 - 6). In addition to the distinctive aspartate cleavage site at P1, the catalytic domains of the caspases require typically four amino acids to the left of the cleavage site with P4 as the prominent specificity-determining residue (9). In contrast to this tetrapeptide specificity, the tri-peptide VAD is able to bind to the active site of every caspase family member studied. Furthermore, addition of a fluoromethyl ketone (FMK) to the tri-peptide results in an irreversible linkage and permanent inactivation of the cysteine protease enzyme (10). Accordingly, the Z-VAD-FMK inhibitor has been shown in numerous studies to effectively inhibit the induction of apoptosis by blocking caspase activation (9, 11). Furthermore, substitution of the amino terminal benzyloxycarbonyl blocking group (Z-) with a detection moiety, such as a fluorescent dye, yields a probe that allows for the detection of caspase activity (12 - 14). FLICA: Fluorescent-Labeled Inhibitors of Caspases The FLICA methodology of caspase detection is available in kit form for assessing individual or poly-caspase activity in cultured cells and tissues. The non-toxic, cell-permeant FLICA reagent enters each cell, where it will irreversibly bind to activated caspases with a preference for its target peptide sequence. For example, active caspase 8 has a high affinity for the peptide sequence L-E-T-D, hence the usage of FAM-LETD-FMK for caspase 8 detection. Because the FLICA reagent FAM-LETD-FMK becomes covalently coupled to the active caspase 8 enzyme, it is retained within the cell during wash steps, while any unbound FLICA reagent diffuses out of the cell and is washed away. The remaining green fluorescent signal is a direct measure of the amount of caspase 8 activity present in the cell at the time the reagent was added. Cells that contain the bound FLICA can be analyzed by 96-well-plate based fluorometry, fluorescence microscopy, or flow cytometry. The carboxyfluorescein (FAM) FLICA reagent has an optimal excitation range from 490 - 495 nm, and emission range from 515 - 525 nm. Cells labeled with the FLICA FAM-LETD-FMK reagent may be read immediately or preserved for 24 hours using the fixative. Unfixed samples may be subsequently analyzed with propidium iodide or Hoechst stain to detect changes in necrosis or nuclear morphology respectively. Other FLICA Caspase Detection Kits, containing the preferred caspase recognition amino acid sequences for poly caspases or caspase 1, 2, 3, 6, 7, 9, 10, and 13, are also available with green or red fluorescence