Novus Caspase Apoptosis Kit, 100 Tests Item ID: NOVNBP229399

Caspase Apoptosis Kit Summary

Description	Assay for apoptosis via poly caspase activation with the red SR FLICA Poly Caspase Assay Kit. This in vitro, whole cell caspase assay employs the red fluorescent caspase inhibitor probe SR-VAD-FMK to label activated caspase enzymes in living cells or tissue samples. Analyze the fluorescent signal in whole cells using fluorescence microscopy, a fluorescent plate reader, or by flow cytometry (green or yellow laser are preferred to detect SR).
Immunogen	FLICA, Fluorescent-Labeled Inhibitor of Caspases, is a simple and accurate method to measure apoptosis via caspase activity in whole cells. Four sample protocols are under the Procedure section.
PreparationMethod	Target: poly caspases Excitation / Emission: 565 nm / 586 nm Method of Analysis: Flow Cytometer, Fluorescence Microscope, Fluorescence Plate Reader Types of Samples: cell culture, tissue
Kit Type	Apoptosis Kit

Packaging, Storage & Formulations

Storage

Store at 4C. Do not freeze.

Applications/Dilutions

Application Notes

The FLICA methodology of caspase detection is available in kit form for assessing individual or poly-caspase activity in cultured cells and tissues. Upon addition to a cultured cell sample, the cell-permeant poly-caspase FLICA SR-VAD-FMK reagent will enter each cell and form irreversible bonds with activated intracellular caspases. Because FLICA reagent becomes covalently coupled to the active 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 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 covalently bound FLICA SR-VAD-FMK poly caspase reagent can be analyzed by 96-well-plate based fluorometry, fluorescence microscopy, or flow cytometry (when a green laser is available). The sulforhofamine (SR) FLICA reagents have an optimal excitation range of 560 - 570 nm, and emission range from 590 - 600 nm. Cells labeled with FLICA may be read immediately or preserved for 24 hours using the Fixative included in the kit. Unfixed samples may be subsequently analyzed with Hoechst stain or 7-AAD to detect changes in nuclear morphology or necrosis, respectively.

Kit Components

Components

10x Apoptosis Wash Buffer 60mL Hoechst 33342 Stain 1mL FLICA Poly Caspases Reagent (SR-VAD-FMK) 4 vials Fixative 6mL

Notes

Adherent Cells Adherent cells need to be carefully washed to avoid the loss of any cells which round up and come off the plate surface. Loose cells may be harvested from the plate or slide surface and treated as suspension cells, while those remaining adherent to the surface should be washed as adherent cells. If the adherent cells are trypsinized, the loose cells can be recombined with the trypsinzed pool, or the washed loose cells can then be recombined with the adherent portion when the analysis is performed. If growing adherent cells on a tissue culture plate, the entire plate may be gently spun as part of the wash process to sediment any loose floating cells. Avoid any attempts to trypsinize cells prior to labeling with a vital dye such as PI. Trypsin exposed cell membranes could become transiently permeant to vital dyes for a variable time period, depending upon the cell line. Cells may be labeled with FLICA before or after trypsinization.

Background

There are two types 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 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).

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Kits are guaranteed for 6 months from date of receipt.