Apoptosis is an evolutionarily conserved form of cell suicide, which follows a specialized cellular process. The central component of this process is a cascade of proteolytic enzymes called caspases. These enzymes participate in a series of reactions that are triggered in response to pro-apoptotic signals and result in the cleavage of protein substrates, causing the disassembly of the cell (Arends & Wyllie 1991).

Caspases have been identified in organisms ranging from C. elegans to humans. The mammalian caspases play distinct roles in apoptosis and inflammation. In apoptosis, caspases are responsible for proteolytic cleavages that lead to cell disassembly (effector caspases), and are involved in upstream regulatory events (initiator caspases). An active caspase consists of two large and two small subunits that form two heterodimers which associate in a tetramer (Slee et al. 1999; Salvesen & Dixit 1997; Alnemri et al. 1996). In common with other proteases, caspases are synthesized as precursors that undergo proteolytic maturation, either autocatalytically or in a cascade by enzymes with similar specificity (Sleath et al. 1990).

Caspase enzymes specifically recognize a 4 or 5 amino acid sequence on the target substrate which necessarily includes an aspartic acid residue. This residue is the target for cleavage, which occurs at the carbonyl end of the aspartic acid residue (Thornberry et al. 1992). Caspases can be detected via immunoprecipitation, immunoblotting techniques using caspase specific antibodies, or by employing fluorochrome substrates which become fluorescent upon cleavage by the caspase.

Test Principle:

CHEMICON′s In Situ Caspase Detection Kits use a novel approach to detect active caspases. The methodology is based on Fluorochrome Inhibitors of Caspases (FLICA). The inhibitors are cell permeable and non-cytotoxic. Once inside the cell, the inhibitor binds covalently to the active caspase (Rotonda et al. 1996). This kit uses a sulforhodamine-labeled fluoromethyl ketone peptide inhibitor of caspase (SR-VAD-FMK), which produces a red fluorescence. When added to a population of cells, the SR-VAD-FMK probe enters each cell and covalently binds to a reactive cysteine residue that resides on the large subunit of the active caspase heterodimer, thereby inhibiting further enzymatic activity. The bound labeled reagent is retained within the cell, while any unbound reagent will diffuse out of the cell and is washed away. The green fluorescent signal is a direct measure of the amount of active caspase present in the cell at the time the reagent was added. Cells that contain the bound labeled reagent can be analyzed by 96-well plate-based fluorometry, fluorescence microscopy, or flow cytometry.

Application:

The CHEMICON In Situ FLICA Pan-Caspase Detection Kit is a fluorescent-based assay for detection of active caspases in cells undergoing apoptosis. The kit is for research use only. Not for use in diagnostic or therapeutic procedures.

· Store unopened kit materials at 2-8°C up to their expiration date. · Reconstituted FLICA Reagent (150X) should be frozen at -20°C for up to 6 months, and may be thawed twice during this time. Aliquot into separate amber tubes if desired. Protect from light at all times. · Store diluted (1X) wash buffer up to -20°C for 2 weeks.

FLICA Reagent (SR-VAD-FMK): Four lyophilized vials 10X Wash Buffer: 60 mL Fixative: 6 mL Hoechst Stain: 1 mL at 200 μg/mL, ready-to-use

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.