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Multiplexing Caspase Activity Detection

The caspase-sensitive peptide fragment-masked amino dyes are digested by a caspase to generate the highly fluorescent dye (or a highly colored dye). The fluorescence (or color) intensity increase is proportional to the caspase activity.
A distinctive feature of the early stages of apoptosis is the activation of caspase enzymes. Members of the caspase (CED-3/ICE) family of cysteine"aspartic acid specific proteases have been identified as crucial mediators of the complex biochemical events associated with apoptosis. The recognition site for caspases is marked by three to four amino acids followed by an aspartic acid residue, with the cleavage occurring after the aspartate. The caspase proteases are typically synthesized as inactive precursors. Inhibitor release or cofactor binding activates the caspases through cleavage at internal aspartates, either by autocatalysis or by the action of another protease.

The normalized fluoresence spectra of AMC, R110 and ProRed™ in aqueous buffer (pH 7.0). AMC, R110 and ProRed™ caspase substrates are well suited for multiplexing caspase activities.
AAT Bioquest offers a diverse selection of capase inhibitors, chromogenic and fluorogenic caspase substrates, and caspase assay kits. Our chromogenic caspase substrates are based on 4-nitroaniline (4-PNA). AAT Bioquest is the only company that offers the multicolor substrates of four distinct fluorescence colors based on 7-Amino-4-methylcoumarin (AMC), 7-Amino-4-trifluoromethylcoumarin (AFC), Rhodamine 110 (R110) and ProRed™ respectively. In particular, the ProRed™-based caspase substrates are extremely useful for screening caspase inhibitors due to their longer excitation and emission wavelengths that eliminate the autofluorescence interference from the compound library.

Detection of caspase activities in Jurkat cells. Jurkat cells were seeded on the same day at 200,000 cells/well in a Costar black wall/clear bottom 96-well plate. The cells were treated with staurosporine at the final concentration of 1 µM for 4 hours (Red Bar) while the untreated cells were used as control (Blue Bar). The fluorescence intensity was measured with FlexStation fluorescence microplate reader at the indicated wavelength. The caspase 3/7, 8 and 9 activities can be detected in a single assay without interferences from other caspases.
AAT Bioquest has developed Cell Meter™ Caspase 3/7, 8 and 9 Activity Multiplexing Assay Kit (Cat# 22820) for multiplexing the detection of caspases 3, 7, 8 and 9. This particular kit is designed to simultaneously monitor four key caspases (caspase 3, 7, 8 and 9) activation involved in cell apoptosis using the three distinct fluorescent colors. This kit uses DEVD-ProRed™, IETD-R110 and LEHD-AMC as fluorogenic indicators for caspase 3/7, 8 and 9 activity respectively. Upon caspase cleavages, DEVD-ProRed™, IETD-R110 and LEHD-AMC caspase substrates generate three distinct fluorophores: ProRed™ (red fluorescence), R110 (green fluorescence) and AMC (blue fluorescence), which can be readily monitored in a single assay due to their nice spectral separation.

Table 1. Multiplexing Caspase Activity and Apoptosis Assay Kits

Cat No.
Product Name
Unit Size
22820Cell Meter™ Multiplexing Caspase 3/7, 8 and 9 Activity Assay Kit *Triple Fluorescence Colors* 100 Tests$547
22850Cell Meter™ Live Cell Caspase 3/7 and Phosphatidylserine Detection Kit *Triple Fluorescence Colors* 100 Tests$327