Imaging Assays for Caspase Activity

Caspases are a family of cysteine–aspartic acid specific proteases (CED-3/ICE) that have been identified as crucial mediators of the complex biochemical events associated with apoptosis (1). Caspases are divided into two groups: initiator and effector caspases, and once activated by the initiator caspase (e.g. caspase-8 and -9), the effector caspases (e.g. caspases-3 and -7) function to cleave protein substrates and initiate apoptosis (2). The recognition site for each caspase 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 and inhibitor release or cofactor binding activates the caspase through cleavage at internal aspartates, either by autocatalysis or by the action of another protease.

Not all caspases are involved with apoptosis and in recent years, it has been demonstrated that some caspases may be involved in inflammation (3). Because cell death cascades are complex and dynamic, it is highly recommended that a multi-parametric approach is conducted for an accurate assessment of apoptosis.

CellEvent Caspase-3/7 detection reagent represents a new generation of caspase substrates and 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 1). This robust 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 2).

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. The newest Caspase-3/7 reagent, CellEvent Caspase-3/7 Red, can also be imaged easily alongside GFP expression in live cells with little to no cross talk (Figure 3).

See caspase assay selection guide

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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 4). 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 (4-8). 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.

See caspase assay selection guide

1. Shalini S, Dorstyn L, Dawar S et al (2015) Cell Death Differ 22: 526–539.
2. Matsuura K, Canfield K, Feng W et al (2016) Int Rev Cell Mol Biol 327: 43–87.
3. Van Opdenbosch N and Lamkanfi M (2019) Immunity 50:1352-1364.
4. Bedner E, Smolewski P, Amstad P et al. (2000) Exp Cell Res 259: 308-313.
5. Amstad PA, Yu G, Johnson GL et al. (2018) BioTechniques 31: 608-616.
6. Dorsey JF, Cunnick JM, Lanehart R et al. (2002) Leukemia 16: 1589–1595.
7. Smolewski P1, Grabarek J, Lee BW et al. (2002) Cytometry 47:143-149.
8. Smolewski P, Grabarek J, Halicka HD et al. (2002) J Immunol Methods 265:111-121.
9. Pozarowski P1, Huang X, Halicka DH, et al. (2003) Cytometry A 55:50-60.