Caspase-9, also known as ICE-LAP6 and Mch6, is an upstream proenzyme in the cascade of enzymatic reactions required to induce cellular apoptosis. After the release of mitochondrial cytochrome c, caspase-9 is activated following its association with the protein complex of Apaf1 and cytochrome c. Active caspase-9 in turn activates pro-caspase-3 promoting the manifestation of some of the more classical features of apoptosis. Activation of caspase-9 can be regulated through protein phosphorylation events. The optimal cleavage recognition sequence for caspase-9 is LEHD. The ability of caspase-9 to associate with both pro-apoptotic and anti-apoptotic proteins as well as its regulatory function during embryonic development suggests that caspase-9 is a key regulator of apoptosis in vivo.

Caspase 9 Activity Assay Kit provides a simple, reproducible, and sensitive tool for measuring the activity of caspase 9 in cell lysate samples. In this assay, caspase 9 cleaves a synthetic specific substrate LEHD, releasing a fluorophore. The fluorescence signal is measured at Ex/Em = 400/505 nm. The increase in fluorescence intensity is directly proportional to caspase 9 activity in the sample.

• Microplate reader capable of measuring fluorescence intensity at Ex/Em = 400/505 nm
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Incubator at 37°C
• Tubes to prepare sample dilutions
• 15 mL and 50 mL conical tubes
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 40 µL of samples to the 96-well microtiter plate. Include substrate background wells by adding Assay Buffer only.
2. Add 10 µL Assay Buffer to each well. If adding an inhibitor, additional Assay Buffer is not needed.
3. Add 100 µL of each standard to the 96-well microtiter plate.
4. Initiate the reaction by adding 50 µL diluted Caspase 9 Substrate solution to each well. Carefully shake the plate for a few seconds to mix well.
5. Measure fluorescence signal. For kinetic reading, immediately start measuring fluorescence intensity at Ex/Em=400/505 nm continuously at 37°C and record data every 5 minutes for 30 minutes.

The entire kit should be stored below -20°C for up to 6 months from the date of shipment.