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You are here:Home » Kits and Assays » Kits-Apoptosis, BioAssay™ » Caspase 8, Fluorometric, BioAssay™ Kit

Caspase 8, Fluorometric, BioAssay™ Kit


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Catalog #C5821
Caspase-8, Fluorometric, BioAssay™ Kit is optimized to detect caspase-8 activity. Enhanced Value It provides enough reagents to perform 100 assays in a 96-well format.
Apoptosis is programmed cell death which leads to the elimination of damaged, unwanted, or unneeded cells without releasing harmful substances into the surrounding area. Apoptosis is involved in a variety of physiological and pathological events,1 ranging from normal fetal development to diseases, such as cancer,2 organ failure and neurodegenerative diseases. Caspases are proteases, which play essential roles in apoptosis and inflammation. There are two types of apoptotic caspases initiator caspases and effector caspases. Caspase-8, an initiator enzyme, also known as Mch5, MACH and FLICE, accepts signals from death receptors (FAS, TRAIL, TNF) and activates downstream effector caspases through proteolytic cleavage.3,4 Activated effector caspases induce apoptosis through cleavage of other cellular substrates such as PARP and DFF.
Caspase-8 has substrate specificity for the amino acid sequence Ile-Glu-Thr-Asp (IETD). The Homogeneous AFC Capase-8 Assay Kit uses Ac-IETD-AFC as the fluorogenic indicator to measure caspase-8 activity. Upon cleavage of the substrate by caspase-8, free AFC (7-amido-4-trifluoromethylcoumarin) emits a yellow-green fluorescence, which can be quantified at excitation/emission=380 nm/500 nm. A bi-functional assay buffer in this kit is optimized for cell lysis and measurement of the enzyme activity. This kit can assay caspase-8 activity in cells grown in a 96-well plate or in larger plates or flasks. It is also adaptable for high throughput screening of apoptosis inducers and inhibitors.
Kit ComponentsComponent A Microtiter Plate 1x100 wells Ac-IETD-AFC, caspase-8 substrate, Ex/Em=380 nm/500 nm upon cleavage 10mM, 1x100ul
Component B AFC (7-amido-4-trifluoromethylcoumarin), fluorescence reference standard, Ex/Em=380nm/500nm 6mM, 1x5ul
Component C Inhibitor of caspase-8, Ac-IETD-CHO 5mM, 1x10ul
Component D Assay Buffer 1x10ml
Component E DTT 1 M, 1x0.5ml
Materials Required (but not provided)• Caspase-8 source: Cell culture, the purified enzyme.
• 96-well microplate: Black tissue culture microplates with clear bottom.
• Fluorescence microplate reader: Capable of detecting emission at 500nm with excitation at 380nm.
Storage and Handling• Store all kit components at -20°C.
• Protect Components A and B from light and from moisture.
• Component D can be stored at room temperature for convenience.
Important NoteThis product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.

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