Description
Description: Detect caspase-9 activity with the red SR-FLICA Caspase 9 Assay Kit. This in vitro assay employs the fluorescent inhibitor probe SR-LEHD-FMK to label active caspase-9 enzymes in living cells or tissue samples. Analyze the fluorescent signal using fluorescence microscopy, a fluorescent plate reader, or by flow cytometry (green or yellow laser required). Covalently Label Active Caspase 9 Whole, Living Cell Assay with Cell-permeant Reagents Counter-stain with other Fluorophores Distinguish Apoptosis from Necrosis 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-9 inhibitor, the enzyme recognition sequence is leucine-glutamic acid-histidine-aspartic acid (LEHD). 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 SR-FLICA reagent is washed away; the remaining red fluorescent signal is a direct measure of caspase-9 activity at the time the probe was added. Detection of nuclear morphology is also possible with the additional kit component, Hoechst 33342, and the cells may be fixed for dual-staining or analysis within 24 hours.
Background: Apoptosis is an evolutionarily conserved form of cell suicide mediated by a cascade of proteolytic enzymes called caspases. 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. Caspase 9 is involved in what is known as the intrinsic apoptosis pathway that results from the mitochondrial release of cytochrome c. The initiator caspase 9 monomer binds other proteins through their caspase activation and recruitment domain (CARD). The initiator caspase -protein interaction results in dimerization of the initiator caspases that leads to their activation. These 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 form of the caspase effector 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). The red FLICA Caspase 9 reagent SR-LEHD-FMK enters each cell and irreversibly binds to activated caspase-9. Because the SR-LEHD-FMK FLICA reagent becomes covalently coupled to the active enzyme, it is retained within the cell, while any unbound SR-LEHD-FMK FLICA reagent diffuses out of the cell and is washed away. The remaining red fluorescent signal is a direct measure of the amount of caspase 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 (with green or yellow laser options). A black microplate like the Costar 96-Well Black Polystyrene Plate is recommended for use with FLICA assays analyzed by a top-reading fluorescence plate reader. The red SR-FLICA reagent has an optimal excitation range from 560 - 565 nm, and emission range from 585 - 595 nm. Cells labeled with the FLICA reagent may be read immediately or preserved for 24 hours using the fixative. Unfixed samples may be subsequently analyzed with Hoechst 33342 stain (included) to observe apoptotic nuclear morphology. Other red SR-FLICA Caspase Assay Kits are available for the detection of poly caspase activity and active caspase 9. FAM-FLICA Caspase Detection Kits with green fluorescent FLICA probes are also available to detect active caspase 1, 2, 3, 6, 8, 9, 10, and 13, as well as for poly-caspase detection