Caspase proteases play an essential role in apoptosis by degrading specific structural, regulatory, and DNA repair proteins within a cell (1, 2). Caspase-9 is activated very early in the apoptotic cascade by cytochrome c, which is released from the mitochondria in response to apoptotic stimuli (3). Activated caspase-9 then initiates the proteolytic activity of other downstream caspases, including caspase-3 and caspase-6. Caspase-8 also initiates the caspase pathway, directly or indirectly activating caspase-3 and other caspases (4, 5).

We offer caspase detection in two formats: assay kits and 96-well plates.

Caspase Assay Kits

The ApoAlert Caspase Assay Kits provide a simple and convenient way to detect caspase protease activity using fluorometric or colorimetric methods. The assay is simple to perform！the kits use crude cell lysates that can be prepared from as few as 1 x 106 adherent cells or cells in suspension. The assay kits detect caspase activation by assaying for the cleavage of a fluorescent substrate (Figure 1) or a colorimetric substrate.For high-throughput analysis, the assay can be performed in a microtiter plate. The entire protocol！from collection of cells to fluorometric or colorimetric results！takes only 90 minutes.

• The Caspase-9/6 Fluorescent Assay detects the shift in fluorescence of 7- amino-4-methoxy coumarin (AMC). Upon cleavage, AMC fluorescence can be measured using a 380-nm excitation filter and a 460-nm emission filter. Fluorescence detection is highly sensitive and can be used to measure even very small amounts of active caspase.
• The Caspase-3 and -8 Fluorescent Assays detect the shift in fluorescence emission of 7-amino-4-trifluoromethyl coumarin (AFC). After the substrate is cleaved by the protease, AFC emits a yellow-green fluorescence at 505 nm if excited at 400 nm.
• The Caspase-3 and -8 Colorimetric Assays measure the proteolytic cleavage of the chromophore p-nitroanilide (pNA) by caspase- 3 or -8. Liberated pNA can be monitored colorimetrically by absorbance at 405 nm.

Caspase Assay Plates

Oftentimes, depending upon the specific cell type as well as the apoptotic stimuli used, one or more caspases will become activated (Figure 2). The ApoAlert Caspase Profiling Assay Plate can be used to simultaneously analyze several different caspases in your sample, with up to 24 wells available for each caspase.

• The ApoAlert Caspase Profiling Plate (Figure 3), measures the activation of four different caspases！caspase-2, -3, -8, and -9！using a fluorometric method in a convenient 96- well plate format. Caspase profiling plates make it easy to profile caspase activities in a variety of cell lines after treatment with different apoptotic stimuli.
• The ApoAlert Caspase-3 Assay Plate provides a convenient way to monitor caspase-3 activity in up to 96 samples at once. No handling of caspase substrates is required, allowing for easy detection of caspase-3 activity in a variety of samples. Caspase-specific inhibitors are included for your control needs.

No Substrate Handling Required

Because the fluorogenic substrates specific for particular caspases are already immobilized in the wells of our 96-well plates, testing a variety of samples is easy, eliminating the need for additional handling of caspase substrates. Upon addition of a cell lysate containing activated caspase to the well, the immobilized fluorogenic substrate is cleaved by the corresponding activated caspase, releasing fluorescent product that can be easily detected by a standard fluorescence plate reader.


Figure 1. Detection of caspase activity during apoptosis. The fluorescent and colorimetric assays are based on the same principle！they detect the fluorophore or chromophore that is cleaved from a substrate by the caspase protease.


Figure 2. Fluorometric detection of caspase activity during apoptosis. After protease activation, the activated caspases recognize their respective substrates which are covalently linked to the fluorogenic dye, 7-amino-4- methyl coumarin (AMC). Upon cleavage by the respective caspase, the free dye can be detected using a plate reader with a 380 nm Excitation and 460 nm Emission filter.