The CaspGLOW™ Fluorescein Active Caspase-9 Staining Kit contains all the reagents necessary to detect active caspase-9 in mammalian cells with high sensitivity. Fluorescein (FITC)-conjugated LEHD-FMK, a specific inhibitor of caspase-9, is utilized in this assay for detection. This reagent is cell permeable, 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.
The caspases constitute a family of aspartate-specific cysteine proteases that mediate a sequence of cleavage events. Recruitment of the inactive proenzyme to oligomerized receptors leads to caspase activation and autoproteolytic cleavage. These active enzymes can then cleave other caspases, thereby generating a caspase signaling cascade that leads to a form of programmed cell death termed apoptosis.
Caspase-9, which is also known as ICE-LAP6 and Mch6, cleaves its substrates at the C-terminal aspartic acid residue of the motif Asp-X-X-Asp. Activation of this signaling protease occurs upon stimulation of death receptors such as CD95/APO-1/Fas, TRAIL-R1, TRAIL-R2, TNFR1, and TRAMP. Caspase-9 is activated by recruitment and dimerization within the Apaf-1 apoptosome. Once recruited, caspase-9 undergoes proteolytic cleavage at Asp315 to yield 35-kDa and 10-kDa fragments. Unlike other caspases, this cleavage event is not required for the catalytic activity of caspase-9. As an initiator caspase, this protease initiates a caspase signaling cascade that results in apoptosis. Substrates of caspase-9 include caspases-3, -6, and -7. Finally, dimerization of cleaved caspase-9 is inhibited by X-linked inhibitor of apoptosis (XIAP).