The Caspase-3/7 Apoptosis Assay Kit provides a simple and reliable method for detecting apoptosis by measuring caspase-3/7 activity. The kit can be used to continuously monitor caspase-3/7 activity in cell extracts and purified enzyme preparations using a fluorometer or fluorescence microplate reader.
The assay employs a rhodamine 110-derived substrate (Z-DEVD-R110), a non-fluorescent bisamide compound. Upon enzymatic cleavage, the substrate is converted in a two-step process: first to a fluorescent monoamide, and then to the more fluorescent R110 product. Both hydrolysis products exhibit spectral properties similar to those of fluorescein, with peak excitation and emission wavelengths of 496 nm and 520 nm, respectively.
In addition to the Z-DEVD-R110 substrate, the kit includes the reversible aldehyde inhibitor Ac-DEVD-CHO and the reference standard R110. The Ac-DEVD-CHO inhibitor allows confirmation that fluorescence signals in both induced and control cell populations arise specifically from caspase-3/7 activity. The reference standard enables quantification of the amount of R110 released during the reaction.
Key Features
• Simple and reliable: Directly measures caspase-3/7 activity for apoptosis detection.
• Continuous monitoring: Tracks activity in cell extracts and purified enzyme preparations.
• Non-fluorescent substrate: Z-DEVD-R110 yields bright fluorescence upon enzymatic cleavage.
• Specificity control: Ac-DEVD-CHO inhibitor confirms signals are caspase-3/7-dependent.
• Quantitative capability: R110 reference standard allows precise product quantification.
Specifications
1. Platform: Microplate Reader, Fluorometer
2. Detection Method: Fluorescent
3. Ex/Em: 496/520 nm
Applications
Caspase activity assay
Components
1. Cell Lysis Buffer: 10 ml
2. Cell Assay Buffer: 10 ml
3. Enzyme Substrate Z-DEVD-R110: 250 µl
4. Enzyme Inhibitor Ac-DEVD-CHO: 20 µl
5. Standard R110: 1 ml
Storage
Store at -20°C and protect from light.
Case Study
Detection of apoptosis activity in Jurkat cells using Caspase-3/7 Apoptosis Assay Kit. Cells were either treated with 10 μM camptothecin for four hours at 37°C to induce apoptosis (induced) or left untreated(control). Both induced and control cells were then harvested, lysed and assayed as described in the kit protocol. Reactions were carried out at room temperature and fluorescence was measured in a fluorescence microplate reader using excitation at 470 ± 10 nm and emission detection at 520 ± 10 nm after the indicated amount of time.
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