Testing Data
(Jurkat cells were treated withstaurosporine, an apoptosis-inducingagent (bottom), or DMSO, a negativecontrol (top), for 4 hours, thenincubated with ICT's green caspase 10inhibitor probe, FAM-AEVD-FMK, for 1hour. Cells were washed twice and readon a flow cytometer. Treatment withstaurosporine induced caspase 10activity in 93.6% of the experimentalcells (M2, bottom right), whereas thenegative control treatment exhibitedcaspase 10 activity in only 4.7% of thecell population (M2, top right). This is a ratio of 20:1. (Dr. Brian W. Lee, ICT).)
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. There are two classes of caspases; 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 also referred to as the extrinsic apoptosis pathway that originates upon activation of cell surface death receptors. Caspases 8 and 10 are monomers that bind to death receptor proteins through their death effector domain (DED) structure. Caspase 9 is also called the intrinsic pathway that esults 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 ubstrate 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 Assay Kits ICT offers many FLICA probes for intracellular caspase detection. The Poly Caspases FLICA reagent FAM-VAD-FMK enters each cell and can irreversibly bind to many activated caspases (caspase-1, -3, -4, -5, -6, -7, -8 and -9). Because the FAM-VAD-FMK FLICA reagent becomes covalently coupled to the active enzyme, it is retained within the cell, 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 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 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 caspase 1, 2, 3, 6, 8, 9, 10, and 13, are also available. In addition to green FLICA probes, we also offer red SR FLICA probes to detect poly-caspases, active caspases 3&7, and active caspase 9.
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Product Notes
The FAM-FLICA (Catalog #AAA258025) is an Assay Kit and is intended for research purposes only. The product is available for immediate purchase. It is sometimes possible for the material contained within the vial of "FAM-FLICA Caspase 10, Assay Kit" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
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