Seeing red during apoptosis

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Cell death mechanisms such as apoptosis are critical for the survival of a multicellular organism. Apoptosis not only ensures proper growth and development by ridding the organism of unneeded cells and tissues, but also minimizes 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 degradation, breakdown of the nucleus, and production of membrane-bound apoptotic bodies [1]. Caspases, a family of cysteine proteases that cleave target proteins at aspartic acid residues, are degradative enzymes that play multiple roles during the initiation and execution of apoptosis. For example, caspase-3 amplifies the signal from initiator caspases such as caspase-8, signifying full commitment to cellular disassembly. In addition to cleaving other caspases in the enzyme cascade, caspase-3 has been shown to cleave poly(ADP-ribose) polymerase (PARP), DNAdependent protein kinase, protein kinase Cδ, and actin.

Introducing the CellEvent Caspase-3/7 Red Reagent

The Invitrogen™ CellEvent™ Caspase-3/7 Red Detection Reagent is a cell-permeant fluorogenic substrate for the detection of activated caspase-3 and -7. Similar to the CellEvent Caspase-3/7 Green Detection Reagent, the CellEvent Caspase-3/7 Red Detection Reagent consists of a 4–amino acid peptide (DEVD) 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 or -7, however, the DEVD peptide is cleaved, enabling the dye to bind to DNA and produce a bright fluorescent signal (Ex/Em = 630/650 nm). The red fluorescence of the dye/DNA complex can be observed using a standard Alexa Fluor™ 647/Cy®5 filter set. Because the cells are alive and have not been lysed during the CellEvent caspase-3/7 assay, they can be simultaneously analyzed for other apoptotic changes such as decreased mitochondrial membrane potential.

The CellEvent Caspase-3/7 Red Reagent is compatible with liveand fixed-cell imaging, high-content analysis (HCA) and high-throughput screening (HTS), and flow cytometry, making it useful for both real-time imaging experiments and endpoint analysis. An important advantage of the CellEvent caspase-3/7 assay is that no wash steps are required for analysis, thus preserving fragile apoptotic cells that are typically lost during these rinses. In addition, 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.

An easy, flexible method for detecting caspase activity

To measure activated caspase-3 or -7 activity, simply add the CellEvent Caspase-3/7 Red Detection Reagent to cells (typically, untreated control cells and cells exposed to an inducer of apoptosis), incubate for 30 minutes, and measure fluorescence. Apoptotic cells with activated caspase-3 or -7 will exhibit bright red-fluorescent nuclei, whereas cells without caspase activity will show minimal fluorescence (Figure 1). Because the cleaved substrate labels nuclei of caspase 3/7–positive cells, the CellEvent caspase-3/7 substrates can also provide information on nuclear morphology, including condensed nuclei typical of late-stage apoptosis. Additionally, the fluorescent signal produced with the CellEvent Red Caspase-3/7 Detection Reagent survives formaldehyde fixation and detergent permeabilization, providing the flexibility to perform endpoint assays and probe for other proteins of interest using immunocytochemical analyses.

In addition to traditional fluorescence microscopy, the CellEvent Caspase-3/7 Red Detection Reagent has been validated for HCA and HTS. The significant difference in fluorescence between normal and apoptotic cells within a population provides an excellent assay window, and the Z´-factor value indicates the reagent is robust enough for use in HTS assays. High-content imaging platforms provide quantitative analysis of individual cells, which can be especially informative when cell responses are not uniform.

Flow cytometry offers an alternative platform for measuring fluorescence of individual cells in a population. Figure 2 shows the flow cytometric analysis of Jurkat cells after treatment with staurosporine to induce apoptosis, followed by staining with the CellEvent Caspase-3/7 Red Flow Cytometry Assay Reagent. As expected, staurosporine-treated cells have a higher percentage of apoptotic cells than the basal levels displayed by the control cells (Figure 2). In addition, using the Invitrogen™ Attune™ NxT Flow Cytometer, we compared the assay performance of the red- and green-fluorescent versions of the CellEvent Caspase-3/7 reagent with staurosporine-treated Jurkat cells and found very good correlation between the dose-response curves for the two reagents (Figure 3).

Figure 1. Detection of caspase-3 and -7 activity using the CellEvent Caspase-3/7 Red Detection Reagent. HeLa cells were seeded into a poly-D-lysine– coated, clear-bottom 96-well microplate in complete medium and incubated overnight to allow for attachment. The next day, cells were treated with (A) DMSO alone or (B) 0.1 μM staurosporine for 4 hr at 37°C. The medium was removed and replaced with PBS + 5% FBS containing 1.62 μM Hoechst™ 33342 (blue) and 5 μM of Invitrogen™ CellEvent™ Caspase-3/7 Red Detection Reagent (red), and cells were incubated for 30 min at 37°C. Images were captured using the Thermo Scientific™ CellInsight™ CX5 High-Content Screening Platform equipped with the 10x objective. Four fields per well of a 96-well microplate were selected for analysis; the calculated Z´ for this assay was 0.45.

Figure 2. Flow cytometric detection of caspase activity in Jurkat cells using the CellEvent Caspase-3/7 Red reagent. Jurkat cells were treated with (A) DMSO or (B) 0.02 μM staurosporine for 4 hr at 37°C before labeling with the CellEvent™ Caspase-3/7 Red Flow Cytometry Assay Reagent. Stained samples were analyzed using the Invitrogen™ Attune™ NxT Flow Cytometer equipped with a 637 nm laser; fluorescence emission was collected with a 670/14 BP filter.

Figure 3. Flow cytometric analysis of caspase-3/7 activity in Jurkat cells using CellEvent Caspase-3/7 detection reagents. Log-phase Jurkat cells were resuspended in complete medium, adjusted to 2 × 106 cells/mL, aliquoted (100 μL/well) into a 96-well, V-bottom microplate, and treated in triplicate with 1 of 8 dilutions of staurosporine for 4 hr at 37°C. Cells were then labeled with 2 μM CellEvent™ Caspase-3/7 Green or Red reagent for 30 min at 37°C and analyzed using the Invitrogen™ Attune™ NxT Flow Cytometer. The BL1 and RL1 channel were used for detection of the CellEvent Caspase-3/7 Green and Red signals, respectively.

Caspase detection to match your experiments

The CellEvent Caspase-3/7 Red Detection Reagent is a robust substrate for measuring caspase-3 and -7 activity using live- or fixed-cell imaging, HTS, or flow cytometry. Together with the CellEvent Caspase-3/7 Green reagent, the CellEvent Caspase-3/7 Red Detection Reagent expands the palette of fluorescence-based caspase assays, allowing more choice when multiplexing with other fluorescent cell structure or function probes (Figure 4).

Figure 4. Multipex detection of apoptosis using the CellEvent Caspase-3/7 Red Detection Reagent and the R-PE Annexin V conjugate. Jurkat cells were treated with (A) DMSO or (B) 10 μM camptothecin for 4 hr at 37°C before labeling with the CellEvent™ Caspase-3/7 Red Flow Cytometry Assay Reagent for 30 min at 37°C. After washing with PBS and Annexin Binding Buffer, the cells were stained with the R-phycoerythrin conjugate of annexin V (R-PE Annexin V) for 15 min at room temperature, washed, and analyzed using the Invitrogen™ Attune™ NxT Flow Cytometer. The numbers in each plot represent the percentage of cell events that fall within each quadrant (labeled Q).

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