Cysteine-dependent aspartate-directed proteases (Caspases) are a family of cysteine proteases that play important roles in apoptosis, necrosis, and inflammation. Sequential activation of caspases plays an important role in cell apoptosis. Caspase-8 is a caspase protein that cleaves and activates Caspase-3 and is involved in the programmed cell death induced by Fas and different apoptotic stimuli. Caspase-8 was detected in the insoluble fraction of the affected brain region in Huntington disease patients, which suggested it plays an important role in neurodegenerative diseases.

Caspase-8 Activity Assay Kit provides a simple, reproducible, and sensitive tool for measuring Caspase-8 activity in cell lysates. This assay is based on spectrophotometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate IETD-pNA. Light emission from pNA can be quantified using a spectrophotometer or a microtiter plate reader at 405 nm. Then, caspase-8 activity can be calculated as nmol of pNA released per minute per milliliter of the sample.

• Microplate reader capable of measuring absorbance at 405 nm
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Tubes to prepare sample dilutions
• Incubator at 37°C
• 15 mL conical tubes
• MilliQ water or another type of double-distilled water (ddH₂O)
• Microplate sealer or plastic wrap

1. Add 50 µL of each sample, control, and background to the 96-well microtiter plate.
2. Add 50 µL of Reaction Buffer with DTT to each well. Mix thoroughly.
3. Add 5 µL of 4 mM Caspase-8 Substrate solution to each well. Mix thoroughly.
4. Incubate the plate for 1 hour at 37°C in the dark with mixing.
5. Measure the absorbance at 405 nm using a plate reader.