CellEvent™ Caspase-3/7 Green Detection Reagent For ApoptosisAlthough the mechanism by which apoptosis is initiated varies depending on cell type and initiating event, several hallmarks of apoptosis can be utilized for detection, including nuclear condensation, plasma membrane blebbing, cleavage of cytoskeletal proteins, and formation of apoptotic bodies . Cysteine aspartic acid proteases, or caspases, are a family of enzymes that play a key role in initiating and executing apoptosis. Activation of caspases is one of the more definitive markers of programmed cell death. The CellEvent™ Caspase-3/7 Green detection reagent is a new fluorogenic substrate for activated caspases 3 and 7 that does not require any wash steps, is compatible with high-content imaging, and is well suited for multiplex imaging in live or fixed cells.
An Easy and Flexible Method for Measuring Activated Caspase 3/7
The CellEvent™ Caspase-3/7 Green detection reagent consists of the DEVD peptide sequence conjugated to a nucleic acid–binding dye. The DEVD peptide inhibits the ability of the dye to bind to DNA and thus the substrate is intrinsically nonfluorescent. In the presence of activated caspase 3/7, the dye is cleaved from the DEVD peptide and is free to bind DNA, producing a fluorogenic response indicative of apoptosis. The fluorescence emission maximum of the dye is approximately 520 nm and can therefore be observed using a standard FITC filter set.
There are several benefits of the CellEvent™ Caspase-3/7 Green detection reagent compared to other methods of detecting activated caspase 3/7. One important advantage of this assay is that no wash steps are required, and thus fragile apoptotic cells typically lost during wash steps are preserved. The CellEvent™ detection reagent is simply added to cells in complete culture medium and incubated for 30 minutes, and samples are ready for imaging. Apoptotic cells with activated caspase 3/7 show bright green nuclei, while cells without activated caspase 3/7 show minimal fluorescence (Figure 1).
This robust assay is highly specific for caspase 3/7 activation and, as expected, nearly complete inhibition of the CellEvent™ Caspase-3/7 Green detection reagent signal was observed after pretreatment of cells with Caspase 3/7 Inhibitor 1 [2,3] (Figure 2). Importantly, the fluorescent signal from the CellEvent™ Caspase-3/7 reagent survives fixation and permeabilization, providing the flexibility to perform end-point assays and probe for other proteins of interest using immunocytochemistry.
Figure 1. No-wash apoptosis detection in live cells. HeLa cells were loaded with 7.5 μM CellEvent™ Caspase-3/7 Green detection reagent and treated with vehicle control (A) or 0.5 μM staurosporine (B) for 4 hr. Untreated cells were also stained with Hoechst 33342 (blue) to visualize the nuclei of negative cells. The fluorescence signal is overlaid with differential interference contrast (DIC). Cells treated with staurosporine show a significant increase in signal from CellEvent™ Caspase-3/7 Green detection reagent (green).
Figure 2. Inhibition of caspase 3/7 activity with a caspase inhibitor. HeLa cells were treated with 0.5 μM staurosporine in the presence of 0–30 μM Caspase 3/7 Inhibitor 1 (EMD Chemicals) for 4 hr. Cells were then labeled with 5 μM CellEvent™ Caspase-3/7 Green detection reagent and Hoechst 33342 (blue) in complete medium. (A) In a dose-dependent manner, the Caspase 3/7 Inhibitor 1 decreased the signal from CellEvent™ Caspase-3/7 Green detection reagent in staurosporine-treated cells, expressed quantitatively as percent positive cells. (B) Visualization of staurosporine-induced caspase 3/7 activity (green); and (C) inhibition thereof is also presented. Images were acquired and analyzed on the Thermo Scientific Cellomics® ArrayScan® VTI platform.
Examine Multiple Parameters of Apoptosis
Apoptosis is commonly evaluated by examining multiple cellular events such as loss of mitochondrial membrane potential, translocation of BAX to mitochondria, translocation of cytochrome c from mitochondria to the cytosol, nuclear condensation, and DNA fragmentation. The CellEvent™ Caspase-3/7 Green detection reagent labels nuclei of caspase 3/7–positive cells to report apoptosis. Therefore, this stain also provides information on nuclear morphology, enabling the detection of two different hallmarks of apoptosis with a single probe.
The CellEvent™ Caspase-3/7 Green detection reagent can also be used with other fluorescent probes in live or fixed cells. Of particular value is the ability of this reagent to report caspase activity in live cells in real time, while simultaneously detecting other parameters related to apoptosis such as changes to mitochondrial membrane potential (Figure 3).
Figure 3. Detecting multiple parameters of apoptosis. HeLa cells were loaded with 50 nM TMRM followed by 5 μM CellEvent™ Caspase-3/7 Green detection reagent. Cells were then treated with 0.5 μM staurosporine. Images were acquired every 5 min over 7 hr at 10x using an ImageXpress® Micro Widefield HCS System (Molecular Devices). Over the 7 hr time course, staurosporine induced a loss of mitochondrial membrane potential followed by activation of caspase 3/7, as indicated by a decrease in TMRM signal (red) and an increase in caspase 3/7 signal (green), respectively.
Suitable for High-Content Screening Assays
The CellEvent™ Caspase-3/7 Green detection reagent can also be used in high-content imaging applications (Figure 4). High-content imaging yields robust quantitative data on populations of cells, which can be extremely informative especially when cellular responses are not uniform. By combining the CellEvent™ Caspase-3/7 reagent with other probes, multiple cellular parameters can be easily examined to define cell death mechanisms or other aspects of cell health.
The CellEvent™ Caspase-3/7 Green detection reagent is a robust reagent for measuring caspase 3/7 activation. Compatible with live- and fixed-cell imaging, this reagent is also amenable to multiplexing, enabling the detection of multiple parameters of apoptosis within the same cell.
Figure 4. High-content imaging and analysis of apoptosis. In two separate experiments, U2OS cells were treated with 30 µM etoposide for 48 hr (A), 0–0.75 µM staurosporine for 24 hr (B), or vehicle control. Cells were then labeled with 7.5 µM CellEvent™ Caspase-3/7 Green detection reagent for 30 min in complete medium at 37°C. (A) Staurosporine treatment resulted in a 16-fold increase in the mean nuclear intensity of treated cells compared to untreated cells. This corresponded to a significant increase in the percentage of cells positive for active caspase 3/7. The Z-factor was calculated based on percentage of positive cells. (B) Dose-response curve and EC50 value for staurosporine in U2OS cells. Cells were analyzed on the Thermo Scientific Cellomics® Arrayscan® VTI platform, and the percentage of cells positive for active caspase 3/7 was determined by thresholding against values for mean nuclear intensity in untreated cells.
Get a copy of this article as it appears in the print version of BioProbes 64.
See a complete listing of the products discussed in this article.