Microscopic view of fibroblasts stained with blue nuclear dye and cellevent caspase probe.

Activation of caspase enzymes, which participate in the cleavage of protein substrates and in the subsequent disassembly of the cell, plays an important role in apoptosis. Thermo Fisher Scientific offers reagents and kits for imaging caspase activity in real-time or as an end-point assay.

What are caspases?

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.

No-wash caspase 3/7 assay for real-time monitoring

CellEvent Caspase-3/7 Green detection reagent represents a new generation of caspase substrates and is an important tool for the study of apoptosis. 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 green-fluorescent signal (absorption/emission maxima ~502/530 nm) 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.

Watch this video of CellEvent Caspase 3/7 Green detection reagent in action:

TMRM and CellEvent Caspase-3/7 Green used to monitor apoptosis progression in HeLa cells treated with staurosporine. HeLa cells were loaded with 50 nM TMRM (red) followed by 5 µM CellEvent Caspase 3/7 Reagent (green). Cells were then treated with 0.5 µM staurosporine and images were acquired every 5 minutes over 7 hours on a Molecular Devices ImageXpress at 10x. Over the 7 hour time course, staurosporine induced a loss of mitochondrial membrane potential followed by activation of caspase 3/7 as evidenced by a decrease in TMRM signal (red) and an increase in caspase 3/7 signal (green), respectively.

Brightfield and fluorescent overlay microscopic image of cells with nuclei stained with blue fluorescence or green fluorescence.

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.

Other caspase assays for end point detection

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 3). 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.

Figure 3. 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.

Citations

Imaging caspase activity selection guide

 CellEvent Caspase-3/7 Detection ReagentImage-iT LIVE Caspase-3 and -7 Detection Kits
ApplicationNo-wash assay to monitor ongoing caspase-3 and -7 activity.End point assay for the detection of active caspase-3 and -7.
ReadoutFollowing interaction with active caspase-3 and -7 in apoptotic cells, the fluorogenic, cell-permeant substrate is able to bind DNA and fluoresce brightly.Fluorescent inhibitor of caspase-3 and -7 interacts with the caspase enzymatic site. Following a wash step, the remaining fluorescence signal is a direct measure of the amount of active caspase at the time the inhibitor was added.
Caspase Target and FunctionCaspases-3 and -7 are downstream effector caspases that, when activated, cleave protein substrates and execute apoptosis
Detection reagentDEVD conjugated to a nucleic acid binding dyeFAM-DEVD-FMKSR-DEVD-FMK
Ex/Em (nm)502/530488/530550/595
Common filterFITCFITCTRITC
Nuclear counterstainsNot included
  • Hoechst 33342
  • propidium iodide
  • Hoechst 33342
  • SYTOX Green
Platform Compatibility
  • Fluorescence microscopy
  • HCS
  • Microplate Reader
  • Fluorescence microscopy
  • HCS
Sample Compatibility
  • Live cells
  • Reagent is fixable
  • Live cells
  • Reagent is fixable
Format25 μL100 μLDropper bottle25 assays25 assays
Cat. No.C10723C10423R37111I35106I35102
 Image-iT LIVE Caspase-8 Detection Kit
ApplicationEnd point assay for the detection of active caspase-8.
ReadoutFluorescent inhibitor of caspases interacts with the caspase-8 enzymatic site. Following a wash step, the remaining fluorescence signal is a direct measure of the amount of active caspase at the time the inhibitor was added.
Caspase Target and FunctionCaspase-8 is an initiator caspase. It functions to activate effector caspases (e.g. caspases-3 and -7).
Detection reagentFAM-LETD-FMK
Ex/Em (nm)488/530
Common filterFITC
Nuclear counterstains
  • Hoechst 33342
  • propidium iodide
Platform Compatibility
  • Fluorescence microscopy
  • HCS
Sample Compatibility
  • Live cells
  • Reagent is fixable
Format25 assays
Cat. No.I35105
 Image-iT LIVE Poly Caspases Detection Kits
ApplicationEnd point assay for the detection of active caspases.
ReadoutFluorescent inhibitor of caspases interacts with the caspase enzymatic site. Following a wash step, the remaining fluorescence signal is a direct measure of the amount of active caspase at the time the inhibitor was added.
Caspase Target and FunctionReagent reacts with multiple caspases (poly-caspases) that bind to the common recognition sequence valine-alanine-aspartic acid (VAD).
Detection reagentFAM-VAD-FMKSR-VAD-FMK
Ex/Em (nm)488/530550/595
Common filterFITCTRITC
Nuclear counterstains
  • Hoechst 33342
  • propidium iodide
  • Hoechst 33342
  • SYTOX Green
Platform Compatibility
  • Fluorescence microscopy
  • HCS
Sample Compatibility
  • Live cells
  • Reagent is fixable
Format25 assays25 assays
Cat. No.I35104I35101
 CellEvent Caspase-3/7 Detection ReagentImage-iT LIVE Caspase-3 and -7 Detection Kits
ApplicationNo-wash assay to monitor ongoing caspase-3 and -7 activity.End point assay for the detection of active caspase-3 and -7.
ReadoutFollowing interaction with active caspase-3 and -7 in apoptotic cells, the fluorogenic, cell-permeant substrate is able to bind DNA and fluoresce brightly.Fluorescent inhibitor of caspase-3 and -7 interacts with the caspase enzymatic site. Following a wash step, the remaining fluorescence signal is a direct measure of the amount of active caspase at the time the inhibitor was added.
Caspase Target and FunctionCaspases-3 and -7 are downstream effector caspases that, when activated, cleave protein substrates and execute apoptosis
Detection reagentDEVD conjugated to a nucleic acid binding dyeFAM-DEVD-FMKSR-DEVD-FMK
Ex/Em (nm)502/530488/530550/595
Common filterFITCFITCTRITC
Nuclear counterstainsNot included
  • Hoechst 33342
  • propidium iodide
  • Hoechst 33342
  • SYTOX Green
Platform Compatibility
  • Fluorescence microscopy
  • HCS
  • Microplate Reader
  • Fluorescence microscopy
  • HCS
Sample Compatibility
  • Live cells
  • Reagent is fixable
  • Live cells
  • Reagent is fixable
Format25 μL100 μLDropper bottle25 assays25 assays
Cat. No.C10723C10423R37111I35106I35102
 Image-iT LIVE Caspase-8 Detection Kit
ApplicationEnd point assay for the detection of active caspase-8.
ReadoutFluorescent inhibitor of caspases interacts with the caspase-8 enzymatic site. Following a wash step, the remaining fluorescence signal is a direct measure of the amount of active caspase at the time the inhibitor was added.
Caspase Target and FunctionCaspase-8 is an initiator caspase. It functions to activate effector caspases (e.g. caspases-3 and -7).
Detection reagentFAM-LETD-FMK
Ex/Em (nm)488/530
Common filterFITC
Nuclear counterstains
  • Hoechst 33342
  • propidium iodide
Platform Compatibility
  • Fluorescence microscopy
  • HCS
Sample Compatibility
  • Live cells
  • Reagent is fixable
Format25 assays
Cat. No.I35105
 Image-iT LIVE Poly Caspases Detection Kits
ApplicationEnd point assay for the detection of active caspases.
ReadoutFluorescent inhibitor of caspases interacts with the caspase enzymatic site. Following a wash step, the remaining fluorescence signal is a direct measure of the amount of active caspase at the time the inhibitor was added.
Caspase Target and FunctionReagent reacts with multiple caspases (poly-caspases) that bind to the common recognition sequence valine-alanine-aspartic acid (VAD).
Detection reagentFAM-VAD-FMKSR-VAD-FMK
Ex/Em (nm)488/530550/595
Common filterFITCTRITC
Nuclear counterstains
  • Hoechst 33342
  • propidium iodide
  • Hoechst 33342
  • SYTOX Green
Platform Compatibility
  • Fluorescence microscopy
  • HCS
Sample Compatibility
  • Live cells
  • Reagent is fixable
Format25 assays25 assays
Cat. No.I35104I35101

Resources

Apoptosis protocols

Fluorescence SpectraViewer
Online tool for visualization of the excitation and emission of fluorescent reagents. Tool allows for checking spectral compatibility for multiple fluorophores.