CellEvent™ Caspase-3/7 Substrate and the Multiplex Analysis of Apoptosis

Bringing Cell Death to Light

Ironically, cell death mechanisms are critical for the survival of a multicellular organism. Apoptosis is essential for proper growth and development by ridding the organism of unneeded cells and tissues, while also minimizing threats by destroying virus-infected or DNA-damaged cells. Cells undergoing apoptosis exhibit an array of morphological and biochemical changes, including decreased mitochondrial membrane potential, loss of plasma membrane asymmetry, protein cleavage and degradation, breakdown of the nucleus, and production of membrane-bound apoptotic bodies [1] (Table 1). Caspases, a family of cysteine aspartic acid proteases, are degradative enzymes that play multiple roles during the initiation and execution of apoptosis. For the study of apoptosis, we offer CellEvent™ Caspase-3/7 Green detection reagent, a fluorogenic substrate for activated caspase-3 and -7. This novel reagent is compatible with high-content imaging and can be multiplexed with other probes for apoptosis and cell health, making it possible to study cell death in the context of other critical cellular functions.

Introducing the CellEvent™ Caspase-3/7 Green Reagent

CellEvent™ Caspase-3/7 Green detection reagent represents a new generation of caspase substrates antd an important tool for the study of apoptosis. The CellEvent™ reagent comprises the DEVD peptide—which contains the recognition site for caspases-3 and -7—conjugated to a nucleic acid–binding dye. Significantly, this dye is fluorescent only when bound to nucleic acids. Because the DEVD peptide inhibits the ability of the dye to bind 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 green-fluorescent signal (fluorescence emission maximum ~520 nm) indicative of apoptosis. 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 Caspase-3/7 Inhibitor 1 [2,3] (data not shown).

No-Wash Imaging with CellEvent™ Caspase-3/7 Green Reagent

An important feature of apoptosis assays with CellEvent™ Caspase-3/7 Green detection reagent 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.

Apoptosis assays with CellEvent™ Caspase-3/7 Green detection reagent are extremely easy to perform. Cells are simply incubated with the CellEvent™ reagent in complete culture medium for 30 minutes and then imaged by traditional fluorescence microscopy (Figure 1) or high-content imaging. Apoptotic cells with activated caspase-3/7 show bright green-fluorescent nuclei, whereas cells without activated caspase-3/7 show minimal fluorescence. Furthermore, the fluorescent signal resulting from cleavage of CellEvent™ Caspase-3/7 Green detection reagent survives fixation and permeabilization, providing the flexibility to perform endpoint assays and to probe for other proteins using immunocytochemical techniques.

Figure 1. Multiplex imaging of apoptosis. U2OS cells were treated with 30 μM etoposide for 18 hr to induce apoptosis. The treated cells were stained first with 7.5 μM CellEvent™ Caspase-3/7 Green detection reagent (green fluorescence) to detect apoptosis and Hoechst 33342 nucleic acid stain (blue fluorescence) to label nuclei, and then with 150 nM MitoTracker® Deep Red FM (pink fluorescence) to label mitochondria. Following fixation and permeabilization, actin was labeled with Alexa Fluor® 546 phalloidin (orange fluorescence).

Multiplex Imaging of Apoptosis and Oxidative Stress

Increased levels of reactive oxygen species (ROS) in cells lead to oxidative stress, which is a key factor in many degenerative diseases. Although pro-oxidants can induce apoptosis and antioxidants can affect its progression, the role of ROS in apoptosis pathways is not yet well defined [4].

Multiplex imaging and analysis can provide a method of examining critical parameters of both apoptosis and oxidative stress. CellEvent™ Caspase-3/7 Green detection reagent can be used together with the CellROX™ Deep Red reagent to follow the temporal progression of apoptosis and oxidative stress, respectively, in a single cell or a cell population (Figure 2). CellROX™ Deep Red reagent is a cell-permeant ROS sensor that is nonfluorescent in its reduced state but produces bright near-infrared fluorescence upon oxidation. The near-infrared emission of the oxidized CellROX™ reagent can be detected simultaneously with the green fluorescence of activated CellEvent™ reagent by traditional fluorescence microscopy as well as by high-content imaging.

Figure 2. Detection of caspase-3/7 activation and ROS in live cells using traditional fluorescence microscopy and high-content imaging. (A) Traditional fluorescence imaging of oxidative stress and apoptosis. HeLa cells were treated with 0.5 µM staurosporine for 0, 2, or 4 hr in the presence of 7.5 µM CellEvent™ Caspase-3/7 Green detection reagent. Cells were then stained with 5 µM CellROX™ Deep Red reagent and Hoechst 33342 nucleic acid stain for 30 min at 37°C, washed with warm DPBS, and imaged immediately. Increased oxidative stress was observed at 2 hr after staurosporine treatment (magenta), whereas caspase-3/7 activation was not observed until 4 hr after treatment (green). (B) High-content analysis revealed the progression of oxidative stress and apoptosis. HeLa cells were treated with 0.5 µM staurosporine for 0–4 hr in the presence of 7.5 µM CellEvent™ Caspase-3/7 Green detection reagent. Cells were then stained with 5 µM CellROX™ Deep Red reagent and Hoechst 33342 stain for 30 min at 37°C, washed with warm DPBS, and analyzed. High-content analysis allowed a more detailed characterization of the increase in ROS levels (red bars) and caspase-3/7 activation (green bars) throughout staurosporine treatment.

No Single Assay Adequately Characterizes Apoptosis

Apoptosis comprises a complex interplay of cellular events, and these events can be selectively targeted with fluorescent probes in order to characterize the spatial and temporal aspects of the pathways (Table 1). Because CellEvent™ Caspase-3/7 Green detection reagent labels the nuclei of caspase-3/7–positive cells, this probe provides information about both caspase-3/7 activation and nuclear morphology.

Moreover, by combining the CellEvent™ reagent with probes that report changes in other cellular functions—such as the loss of mitochondrial membrane potential, the translocation of phosphatidylserine to the outer membrane, and DNA fragmentation—you can obtain a more complete picture of the state of the apoptotic cell over time.

Study Apoptosis in Context

As with cell viability, no single parameter fully defines cell death in all systems. Therefore, it is often advantageous to use a multiparametric approach when studying apoptosis and other cell death pathways.

Table 1. Representative time course of apoptosis, using camptothecin-induced Jurkat cells as a model system.*

References

1. Taylor RC, Cullen SP, Martin SJ (2008) Nat Rev Mol Cell Biol 9:231–241.
2. Lee D, Long SA, Murray JH et al. (2001) J Med Chem 44:2015–2026.
3. Nicholson DW (1999) Cell Death Differ 6:1028–1042.
4. Avery SV (2011) Biochem J 434:201–210.

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.