Description
Description: Forget the lysates. Assay for apoptosis via caspase-3 activity in whole, living cells with the FLICA 660 Caspase-3 Assay Kit. - Ex: 660 nm / Em: 680-690 nm - Whole cell analysis via flow cytometry or fluorescence microscopy - Flexible multiparametric analysis with additional dyes or probes - Compatibility with GFP and green autofluorescence - Benchtop flow compatible This new caspase-3 assay for in vitro apoptosis detection employs a new far-red fluorescent caspase-3 and -7 inhibitor probe, FLICA 660-DEVD-FMK, to label active caspase-3 and-7 enzymes in living cells or tissue samples. Image the fluorescent signal using fluorescence microscopy or analyze samples for caspase-3 and -7 activity by flow cytometry.
Background: Caspase-3 Assay for Apoptosis Detection in Whole, Living Cells (25-50 tests) 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. 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 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. These preferred tetra-peptide sequences have been used to derive peptides that specifically compete for caspase binding. In addition to the distinctive aspartate cleavage site at P1, the catalytic domains of the caspases typically require four amino acids to the left of the cleavage site with P4 as the prominent specificity-determining residue. Addition of a fluoromethyl ketone (FMK) to the tetrapeptide results in an irreversible linkage and permanent inactivation of the cysteine protease enzyme. Furthermore, conjugation of a fluorescent moiety at the amino terminus yields a probe that allows for the detection of caspase activity. FLICA 660 Caspase-3 Assay: Detection Mechanism The far-red FLICA 660-DEVD-FMK caspase-3 detection probe is comprised of the preferred affinity peptide sequence (DEVD) targeted by activated caspase-3 and caspase-7, a far-red fluorescent 660 dye label, and a fluoromethyl ketone (FMK) reactive moiety. The resulting fluorescent caspase-3 inhibitor probe forms an irreversible, covalent bond with active caspase-3 enzymes, efficiently labeling the target for detection. Due to its cell permeant nature and fluorescence properties, the far-red FLICA caspase-3 detection probe enables whole cell analysis via common fluorescence detection methods. To use FLICA Caspase-3 Assay, add the caspase-3 detection probe directly to suspension cell or tissue culture media, incubate, and wash. The cell permeant, far-red FLICA caspase-3 detection probe will efficiently diffuse into cells and irreversibly bind to activated caspase-3 enzymes, thereby retaining the red signal inside caspase-3-positive cells. Cells not bearing active caspase-3 return to a non-fluorescent status after the wash step. The FLICA 660 caspase-3 detection probe has an optimal excitation at 660 nm and optimal emission range from 680-690 nm. As such, it has demonstrated excellent excitation efficiency with a conventional red HeNe laser with a 633 nm excitation, enabling samples to be analyzed with most flow cytometers and fluorescence microscopes equipped with electronic grey scale image capabilities. Cells labeled with the FLICA caspase-3 detection probe may be read immediately or preserved for 16 hours using the fixative. Additional FLICA caspase assays are in development