The CaspGLOW Fluorescein Staining Kit contains reagents to sensitively detect active caspase-3 in mammalian cells. This assay uses the specific Caspase-3 inhibitor, DEVD-FMK, which is directly conjugated to FITC (fluorescein isothiocyanate) for the detection system. This reagent is permeable to cells, non-toxic, and irreversibly binds to the active enzyme. Detection of the labeled cells can be determined by flow cytometry, fluorescent microscopy, or a fluorescent plate reader.

Caspases are a family of aspartate-specific cysteine ​​proteases that act in a staggered fashion like kinases. Recruitment of these proteases into oligomerized receptors leads to autoproteolytic activation and cleavage. Active caspases can proteolyze additional caspases generating a caspase cascade. The end result of this signaling pathway is a form of controlled cell death, called apoptosis.

Caspase-3, also known as Yama, CPP32, and apopain, cleaves their substrates at the carboxyl end of aspartate residues. Active caspase-3 consists of 2 sets of homodimers (~ 17 and 12 kDa) that are derived from two caspase-3 precursor polypeptides and have two active sites. Caspase-3 is proteolytically activated by other caspases.

Both subunits contribute to substrate binding and catalysis. The active site cysteine ​​that is covalently bound to the substrate is near the C-terminus of the large subunit. Active Caspase-3 has a double symmetry, two active site pockets, each of which resides on an opposite side. Caspase-3, along with caspases 8 and 9, is located at the fundamental junctions of apoptotic pathways. Caspase-3 appears to amplify the initiation signals of Caspase 8 and 9 in a complete compromise with apoptotic disassembly.

Reported Application

Flow cytometric analysis, immunocytochemistry

Caspase activation plays a central role in apoptosis. The CaspGLOW  Fluorescein Active Caspase-3 Staining Kit provides a convenient means for the sensitive detection of activated caspase-3 in living cells.

The assay uses the caspase-3 inhibitor, DEVD-FMK, conjugated to FITC (FITC-DEVD-FMK) as a marker. FITC-DEVD-FMK is cell-permeable, non-toxic, and irreversibly binds to activated caspase-3 in apoptotic cells.

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