Caspase Assays

CellEvent Caspase-3/7 is easy to use. Simply prepare fresh CellEvent Caspase-3/7 staining solution, then add directly to your cells, incubate for 30–60 minutes, and then visualize your cells (Figure 1). Apoptotic cells will have activated caspase-3/7 resulting in bright green or red nuclei, while cells without activated caspase-3/7 (non-apoptotic cells) will have minimal fluorescent signal.

CellEvent Caspase-3/7 detection reagent is an important tool for the study of apoptosis. It is available in both green (Ex/Em: 502/530) and red (Ex/Em: 590/610) and can be visualized with a FITC or Texas Red filter (respectively) on any fluorescence microscope. The cell-permeant CellEvent reagent comprises the four–amino acid peptide DEVD—which contains the recognition site for caspases 3 and 7—conjugated to a nucleic acid–binding dye. Because the DEVD peptide inhibits the ability of the dye to bind to DNA, CellEvent Caspase-3/7 Green detection reagent is intrinsically nonfluorescent. In the presence of activated caspase 3/7, the dye is cleaved from the DEVD peptide and free to bind DNA, producing a bright fluorescent signal indicative of apoptosis (Figure 2). This caspase assay is highly specific for caspase 3/7 activation and, as expected, we observe nearly complete inhibition of the CellEvent Caspase-3/7 Green detection reagent signal in cells pretreated with a caspase 3/7 inhibitor (Figure 3).

One important advantage of the CellEvent Caspase-3/7 assay is that no wash steps are required, thus preserving fragile apoptotic cells that are typically lost during these rinses. The loss of apoptotic cells during wash steps may lead to an underestimation of the extent of apoptosis in the sample, resulting in poor assay accuracy. Additionally, the fluorescent signal resulting from cleavage of CellEvent Caspase-3/7 detection reagent survives formaldehyde fixation and detergent permeabilization, providing the flexibility to perform endpoint assays and to probe for other proteins using immunocytochemical techniques. CellEvent Caspase-3/7 Red, can also be imaged easily alongside GFP expression in live cells with little to no cross talk (Figure 4).

The Image-iT LIVE Caspase Detection Kits employ a novel approach to detect active caspases that is based on a fluorescent inhibitor of caspases (FLICA) methodology, essentially an affinity label. The reagent associates a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, with a caspase-specific amino acid sequence. For the caspase-3 and -7 reagents, this recognition sequence is aspartic acid-glutamic acid- valine-aspartic acid (DEVD); for the poly caspases reagents, this recognition sequence is valine-alanine-aspartic acid (VAD) and for caspase-8 reagent this recognition sequence is leucine-glutamic acid-threonine-aspartic acid (LETD). A fluorescent dye is attached as a reporter. The FLICA reagent is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently through the FMK moiety. The FLICA inhibitor is cell permeant and noncytotoxic. Unbound FLICA molecules diffuse out of the cell and are washed away; the remaining fluorescent signal is a direct measure of the amount of active caspase that was present at the time the inhibitor was added (Figure 5). Each kit contains a cell-impermeant nucleic acid stain to monitor plasma membrane integrity.

FLICA reagents have been used widely to study apoptosis using flow cytometry and microscopy (7, 8). However it should be noted that while cellular fluorescence from the reagent is strongly linked to caspase activity in apoptotic cells, its interaction with other cellular sites may contribute to signal intensity in non-apoptotic cells (9). Appropriate controls should be included in any experimental design.

Figure 5. Live HeLa cells visualized using the Image-iT LIVE Red Poly Caspase Detection Kit. Live HeLa cells visualized using the Image-iT LIVE Red Poly Caspase Detection Kit (I35101). Cells were treated with staurosporine to induce apoptosis, and FLICA reagent (SR-VAD-FMK, left) was added. Cells were then loaded with SYTOX Green dye (middle) and Hoechst 33342 (blue fluorescence, right) and analyzed by fluorescence microscopy. Apoptotic, non-apoptotic, and dead cells are all visible in the same field.

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The CellEvent Caspase-3/7 Green flow cytometry assay allows for the detection of activated caspase-3 and caspase-7 in apoptotic cells. Cell-permeant CellEvent Caspase-3/7 detection reagent consists of a four-amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye. During apoptosis, caspase-3 and caspase-7 proteins are activated and able to cleave the caspase 3/7 recognition sequence encoded in the DEVD peptide. Cleavage of the recognition sequence and binding of DNA by the reagent labels the apoptotic cells with a bright, fluorogenic signal. When used together with the appropriate SYTOX dead cell stain, apoptotic cells can be easily discriminated from live and necrotic cells (Figure 6).

Figure 6. Caspase activity detection in Jurkat cells using the CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit on the Attune Flow Cytometer. Jurkat cells (T-cell leukemia, human) were treated with (A) DMSO or (B) 10 µM camptothecin for 3 hours before labeling with the CellEvent Caspase 3/7 Green Flow Cytometry kit. Stained samples were analyzed on the Attune Acoustic Focusing Cytometer equipped with a 488-nm laser, and fluorescence emission was collected using a 530/30 BP filter for CellEvent Caspase 3/7 Green Detection Reagent and a 690/50BP filter for SYTOX AADvanced stain, respectively. Note that the treated cells have a higher percentage of apoptotic cells (panel B) than the basal level of apoptosis seen in the control cells (panel A). A = apoptotic cells, V = viable cells, N = necrotic cells.

The Vybrant FAM Caspase Assay Kits, also known as the Vybrant FLICA Caspase Apoptosis Assay Kits, are easy to use and do not require the need for special buffers to lyse or permeabilize the cell membranes. Simply add the reagent to your cell culture media, let it incubate 1–4 hours, wash the cells, and analyze (Figure 7).

These assays employ the fluorescent inhibitor of caspases (FLICA) methodology. The FLICA reagent, a caspase inhibitor, binds covalently and irreversibly to the reactive cysteines of active caspases. This binding inhibits further enzymatic activity and makes this an end point assay for caspase activity (Figure 8). Vybrant FAM Caspase Assay Kits allow for the accurate detection of active caspases in living cells, are more reliable than annexin V, and simpler to use than TUNEL assays.