Figure 8. Detection of active caspases in living cells by flow cytometry. Jurkat cells were either treated with 10 μM camptothecin for 4 hours at 37ºC and 5% CO2 and stained with the FLICA reagent for caspase-3 and -7 and propidium iodide, both from the Vybrant FAM Caspase-3 and -7 Assay Kit. Samples were analyzed on a flow cytometer with 488 nm excitation using 530 nm bandpass and 670 nm longpass emission filters.
Caspases, key regulators of apoptosis, play an important role in programmed cell death. Caspase enzymes are responsible for cleaving protein substrates and executing the dismantling of cellular components during apoptosis. Thermo Fisher Scientific offers reagents and kits for detecting caspase activity in real-time or as end-point assays. Real-time caspase activity assays are an easy-to-use, no-wash method that allow for the visualization and monitoring of caspase activity in live cells. End-point caspase activity assays offer a reliable and convenient method for quantifying caspase activity at a specific time point. Our assays enable researchers to gain valuable insights into the mechanisms underlying cell death processes.
What are caspases?
Caspases are a family of protease enzymes that are crucial mediators of programmed cell death or apoptosis. They are divided into two groups: initiator caspases (e.g., caspase-8 and -9) and executioner caspases (e.g., caspase-3 and -7). Initiator caspases activate executioner caspases, which then function to cleave structural proteins and activate other enzymes (1, 2).
Caspase proteases are synthesized as inactive precursors, also known as procaspases. Activation of initiator caspases occurs by dimerization, induced proximity, or association with specific binding partners. Executioner caspases are then activated by these initiator caspases or other activated executioner caspases. These executioner caspases then cleave specific proteins to start the cascade of events involved in cell death (3).
Caspases are not only involved in apoptosis but have been demonstrated to be involved in non-cell death processes such as inflammation, dendrite trimming, and cell differentiation (4). Dysregulation of caspases has been shown to contribute to various pathological conditions and human disease such as tumorigenesis, autoimmunity, autoinflammation, cancer, and neurodegenerative disorders (5,6).
Caspase assays
Caspase assays are designed to detect and quantify caspase activity within cells, providing valuable insights into the apoptotic process. The use of specific substrates allows researchers to measure the enzymatic activity of caspases evaluating apoptosis over time or at a specific time. Since cell death cascades are complex and dynamic, it is highly recommended that a multi-parametric approach is conducted for an accurate assessment of apoptosis.
Learn about alternative apoptosis assays
CellEvent Caspase-3/7 Green | CellEvent Caspase-3/7 Red | CellEvent Caspase-3/7 Green and Red | CellEvent Caspase-3/7 Green ReadyProbes Reagent | |||
|---|---|---|---|---|---|---|
| Application | No-wash assay to monitor ongoing caspase-3/7 activity. | |||||
| Readout | Following interaction with active caspase-3/7 in apoptotic cells, the fluorogenic, cell-permeant substrate is able to bind DNA and fluoresce brightly. | |||||
| Caspase target and function | Caspases-3 and -7 are downstream effector caspases that, when activated, cleave protein substrates and execute apoptosis. | |||||
| Detection reagent | DEVD conjugated to a nucleic acid binding dye | |||||
| Ex/Em (nm) | 502/530 | 590/610 | 502/530 | 590/610 | 502/530 | |
| Suggested filter | FITC | Texas Red | FITC | Texas Red | FITC | |
| Instrument platform |
|
| ||||
| Sample compatibility |
|
| ||||
| Format | various | 1 vial (lyophilized powder)* or 5 vials (lyophilized powder) | 2 vials (1 Green, 1 Red) | Dropper bottle | ||
| See all CellEvent Caspase-3/7 Detection Reagents | R37111 | |||||
| *1 vial=100 assays using 96-well plate; **5 vials=500 assays using 96-well plate | ||||||
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.
How do you use CellEvent Caspase-3/7?
CellEvent Caspase-3/7 is easy to use. Simply prepare fresh CellEvent Caspase-3/7 staining solution, then add directly to your cells, incubate for 30–60 minutes, and then visualize your cells (Figure 1). Apoptotic cells will have activated caspase-3/7 resulting in bright green or red nuclei, while cells without activated caspase-3/7 (non-apoptotic cells) will have minimal fluorescent signal.
Figure 1. Schematic of CellEvent Caspase-3/7 protocol.
No-wash caspase-3/7 assay for real-time monitoring
CellEvent Caspase-3/7 detection reagent 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 2). This caspase 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 3).
Figure 2. 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 3. 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.
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. CellEvent Caspase-3/7 Red, can also be imaged easily alongside GFP expression in live cells with little to no cross talk (Figure 4).
Figure 4. Expression of GFP with Caspase Red shows minimal crosstalk. A673 and U2OS cells were plated at 3,000 cells per well and transduced with cytosolic GFP BacMam and treated with DMSO or 2 µM camptothecin for 24 hours at 37°C. Cells were subsequently stained with CellEvent Caspase 3/7 Red for 1 hour at 37°C prior to imaging on an EVOS M7000 widefield fluorescence microscope.
| Image-iT LIVE Green Caspase-3 and -7 Detection Kit | Image-iT LIVE Red Caspase-3 and -7 Detection Kit | Image-iT LIVE Green Poly Caspase Detection Kit | Image-iT LIVE Red Poly Caspase Detection Kit | |
|---|---|---|---|---|
| Application | End point assay for the detection of active caspases. | |||
| Readout | Fluorescent 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 function | Image-iT LIVE Green and Red Caspase-3 and -7 detect caspases-3 and -7, downstream effector caspases that, when activated, cleave protein substrates and execute apoptosis. | Image-iT LIVE Green and Red Poly Caspase reagent reacts with multiple caspases (poly-caspases) that bind to the common recognition sequence valine-alanine-aspartic acid (VAD). | ||
| Detection reagent | FAM-DEVD-FMK | SR-DEVD-FMK | FAM-VAD-FMK | SR-VAD-FMK |
| Ex/Em (nm) | 488/530 | 550/595 | 488/530 | 550/595 |
| Suggested filters | FITC | TRITC | FITC | TRITC |
Nuclear counterstains | Hoechst 33342 Propidium iodide | Hoechst 33342 SYTOX Green | Hoechst 33342 Propidium iodide | Hoechst 33342 SYTOX Green |
Instrument platform | Fluorescence microscopy HCS | Fluorescence microscopy HCS | ||
| Sample compatibility | Live cells Reagent is fixable | Live cells Reagent is fixable | ||
| Format | 25 assays | 25 assays | ||
| See all Image-iT LIVE Red and Green Caspase Detection Kits | ||||
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 5). 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 (7, 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 5. 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.
CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit | Vybrant FAM Poly Caspases Assay Kit | Vybrant FAM Caspase-3 and -7 Assay Kit | Vybrant FAM Caspase-8 Assay Kit | |
|---|---|---|---|---|
| Application | No-wash assay to monitor ongoing caspase-3/7 activity. | End point assay for the detection of caspase activity. | ||
| Readout | Following interaction with active caspase-3/7, the fluorogenic, cell-permeant substrate is able to bind DNA and fluoresce brightly. | Fluorescent 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 function | Detects caspase-3 and -7, downstream effector caspases that, when activated, cleave protein substrates and execute apoptosis. | Reacts with multiple caspases (poly-caspases) that bind common recognition sequence valine-alanine-aspartic acid (VAD). | Reacts with caspase-3 and -7 that bind common recognition sequence aspartic acid-glutamic acid-valine-aspartic acid (DEVD). | Reacts caspase-8 that bind common recognition sequence leucine-glutamic acid-threonine-aspartic acid (LETD). |
| Detection reagent | DEVD conjugated to a nucleic acid binding dye | FAM-VAD-FMK | FAM-DEVD-FMK | FAM-LETD-FMK |
| Ex/Em (nm) | 511/533 | 495/529 | 388/530 | 488/530 |
| Laser (nm) | 488 | 488 | ||
| Nuclear counterstain | SYTOX AADvanced | Hoechst 33342 Propidium iodide | ||
| Sample compatibility | Live cells | Live cells | ||
| Format | 20 assays 100 assays | 25 assays | ||
| See all CellEvent Caspase-3/7 Green Assay Kits | See all Vybrant FLICA Caspase Apoptosis Assay Kits | |||
Real-time, no-wash caspase-3/7 assay for flow cytometry
The CellEvent Caspase-3/7 Green flow cytometry assay allows for the detection of activated caspase-3 and caspase-7 in apoptotic cells. Cell-permeant CellEvent Caspase-3/7 detection reagent consists of a four-amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye. During apoptosis, caspase-3 and caspase-7 proteins are activated and able to cleave the caspase 3/7 recognition sequence encoded in the DEVD peptide. Cleavage of the recognition sequence and binding of DNA by the reagent labels the apoptotic cells with a bright, fluorogenic signal. When used together with the appropriate SYTOX dead cell stain, apoptotic cells can be easily discriminated from live and necrotic cells (Figure 6).
Figure 6. Caspase activity detection in Jurkat cells using the CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit on the Attune Flow Cytometer. Jurkat cells (T-cell leukemia, human) were treated with (A) DMSO or (B) 10 µM camptothecin for 3 hours before labeling with the CellEvent Caspase 3/7 Green Flow Cytometry kit. Stained samples were analyzed on the Attune Acoustic Focusing Cytometer equipped with a 488-nm laser, and fluorescence emission was collected using a 530/30 BP filter for CellEvent Caspase 3/7 Green Detection Reagent and a 690/50BP filter for SYTOX AADvanced stain, respectively. Note that the treated cells have a higher percentage of apoptotic cells (panel B) than the basal level of apoptosis seen in the control cells (panel A). A = apoptotic cells, V = viable cells, N = necrotic cells.
End-point caspase assays for flow cytometry
The Vybrant FAM Caspase Assay Kits, also known as the Vybrant FLICA Caspase Apoptosis Assay Kits, are easy to use and do not require the need for special buffers to lyse or permeabilize the cell membranes. Simply add the reagent to your cell culture media, let it incubate 1–4 hours, wash the cells, and analyze (Figure 7).
Figure 7.Detecting active caspases in apoptotic cells using the Vybrant FAM Caspase Assay Kits. Unbound FLICA reagent diffuses out of the cell and is washed away. The green-fluorescent signal is a direct measure of the amount of active caspase that was present at the time the inhibitor was added.
These assays employ the fluorescent inhibitor of caspases (FLICA) methodology. The FLICA reagent, a caspase inhibitor, binds covalently and irreversibly to the reactive cysteines of active caspases. This binding inhibits further enzymatic activity and makes this an end point assay for caspase activity (Figure 8). Vybrant FAM Caspase Assay Kits allow for the accurate detection of active caspases in living cells, are more reliable than annexin V, and simpler to use than TUNEL assays.
What are caspases?
Caspases are a family of protease enzymes that are crucial mediators of programmed cell death or apoptosis. They are divided into two groups: initiator caspases (e.g., caspase-8 and -9) and executioner caspases (e.g., caspase-3 and -7). Initiator caspases activate executioner caspases, which then function to cleave structural proteins and activate other enzymes (1, 2).
Caspase proteases are synthesized as inactive precursors, also known as procaspases. Activation of initiator caspases occurs by dimerization, induced proximity, or association with specific binding partners. Executioner caspases are then activated by these initiator caspases or other activated executioner caspases. These executioner caspases then cleave specific proteins to start the cascade of events involved in cell death (3).
Caspases are not only involved in apoptosis but have been demonstrated to be involved in non-cell death processes such as inflammation, dendrite trimming, and cell differentiation (4). Dysregulation of caspases has been shown to contribute to various pathological conditions and human disease such as tumorigenesis, autoimmunity, autoinflammation, cancer, and neurodegenerative disorders (5,6).
Caspase assays
Caspase assays are designed to detect and quantify caspase activity within cells, providing valuable insights into the apoptotic process. The use of specific substrates allows researchers to measure the enzymatic activity of caspases evaluating apoptosis over time or at a specific time. Since cell death cascades are complex and dynamic, it is highly recommended that a multi-parametric approach is conducted for an accurate assessment of apoptosis.
Learn about alternative apoptosis assays
CellEvent Caspase-3/7 Green | CellEvent Caspase-3/7 Red | CellEvent Caspase-3/7 Green and Red | CellEvent Caspase-3/7 Green ReadyProbes Reagent | |||
|---|---|---|---|---|---|---|
| Application | No-wash assay to monitor ongoing caspase-3/7 activity. | |||||
| Readout | Following interaction with active caspase-3/7 in apoptotic cells, the fluorogenic, cell-permeant substrate is able to bind DNA and fluoresce brightly. | |||||
| Caspase target and function | Caspases-3 and -7 are downstream effector caspases that, when activated, cleave protein substrates and execute apoptosis. | |||||
| Detection reagent | DEVD conjugated to a nucleic acid binding dye | |||||
| Ex/Em (nm) | 502/530 | 590/610 | 502/530 | 590/610 | 502/530 | |
| Suggested filter | FITC | Texas Red | FITC | Texas Red | FITC | |
| Instrument platform |
|
| ||||
| Sample compatibility |
|
| ||||
| Format | various | 1 vial (lyophilized powder)* or 5 vials (lyophilized powder) | 2 vials (1 Green, 1 Red) | Dropper bottle | ||
| See all CellEvent Caspase-3/7 Detection Reagents | R37111 | |||||
| *1 vial=100 assays using 96-well plate; **5 vials=500 assays using 96-well plate | ||||||
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.
How do you use CellEvent Caspase-3/7?
CellEvent Caspase-3/7 is easy to use. Simply prepare fresh CellEvent Caspase-3/7 staining solution, then add directly to your cells, incubate for 30–60 minutes, and then visualize your cells (Figure 1). Apoptotic cells will have activated caspase-3/7 resulting in bright green or red nuclei, while cells without activated caspase-3/7 (non-apoptotic cells) will have minimal fluorescent signal.
Figure 1. Schematic of CellEvent Caspase-3/7 protocol.
No-wash caspase-3/7 assay for real-time monitoring
CellEvent Caspase-3/7 detection reagent 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 2). This caspase 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 3).
Figure 2. 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 3. 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.
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. CellEvent Caspase-3/7 Red, can also be imaged easily alongside GFP expression in live cells with little to no cross talk (Figure 4).
Figure 4. Expression of GFP with Caspase Red shows minimal crosstalk. A673 and U2OS cells were plated at 3,000 cells per well and transduced with cytosolic GFP BacMam and treated with DMSO or 2 µM camptothecin for 24 hours at 37°C. Cells were subsequently stained with CellEvent Caspase 3/7 Red for 1 hour at 37°C prior to imaging on an EVOS M7000 widefield fluorescence microscope.
| Image-iT LIVE Green Caspase-3 and -7 Detection Kit | Image-iT LIVE Red Caspase-3 and -7 Detection Kit | Image-iT LIVE Green Poly Caspase Detection Kit | Image-iT LIVE Red Poly Caspase Detection Kit | |
|---|---|---|---|---|
| Application | End point assay for the detection of active caspases. | |||
| Readout | Fluorescent 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 function | Image-iT LIVE Green and Red Caspase-3 and -7 detect caspases-3 and -7, downstream effector caspases that, when activated, cleave protein substrates and execute apoptosis. | Image-iT LIVE Green and Red Poly Caspase reagent reacts with multiple caspases (poly-caspases) that bind to the common recognition sequence valine-alanine-aspartic acid (VAD). | ||
| Detection reagent | FAM-DEVD-FMK | SR-DEVD-FMK | FAM-VAD-FMK | SR-VAD-FMK |
| Ex/Em (nm) | 488/530 | 550/595 | 488/530 | 550/595 |
| Suggested filters | FITC | TRITC | FITC | TRITC |
Nuclear counterstains | Hoechst 33342 Propidium iodide | Hoechst 33342 SYTOX Green | Hoechst 33342 Propidium iodide | Hoechst 33342 SYTOX Green |
Instrument platform | Fluorescence microscopy HCS | Fluorescence microscopy HCS | ||
| Sample compatibility | Live cells Reagent is fixable | Live cells Reagent is fixable | ||
| Format | 25 assays | 25 assays | ||
| See all Image-iT LIVE Red and Green Caspase Detection Kits | ||||
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 5). 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 (7, 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 5. 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.
CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit | Vybrant FAM Poly Caspases Assay Kit | Vybrant FAM Caspase-3 and -7 Assay Kit | Vybrant FAM Caspase-8 Assay Kit | |
|---|---|---|---|---|
| Application | No-wash assay to monitor ongoing caspase-3/7 activity. | End point assay for the detection of caspase activity. | ||
| Readout | Following interaction with active caspase-3/7, the fluorogenic, cell-permeant substrate is able to bind DNA and fluoresce brightly. | Fluorescent 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 function | Detects caspase-3 and -7, downstream effector caspases that, when activated, cleave protein substrates and execute apoptosis. | Reacts with multiple caspases (poly-caspases) that bind common recognition sequence valine-alanine-aspartic acid (VAD). | Reacts with caspase-3 and -7 that bind common recognition sequence aspartic acid-glutamic acid-valine-aspartic acid (DEVD). | Reacts caspase-8 that bind common recognition sequence leucine-glutamic acid-threonine-aspartic acid (LETD). |
| Detection reagent | DEVD conjugated to a nucleic acid binding dye | FAM-VAD-FMK | FAM-DEVD-FMK | FAM-LETD-FMK |
| Ex/Em (nm) | 511/533 | 495/529 | 388/530 | 488/530 |
| Laser (nm) | 488 | 488 | ||
| Nuclear counterstain | SYTOX AADvanced | Hoechst 33342 Propidium iodide | ||
| Sample compatibility | Live cells | Live cells | ||
| Format | 20 assays 100 assays | 25 assays | ||
| See all CellEvent Caspase-3/7 Green Assay Kits | See all Vybrant FLICA Caspase Apoptosis Assay Kits | |||
Real-time, no-wash caspase-3/7 assay for flow cytometry
The CellEvent Caspase-3/7 Green flow cytometry assay allows for the detection of activated caspase-3 and caspase-7 in apoptotic cells. Cell-permeant CellEvent Caspase-3/7 detection reagent consists of a four-amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye. During apoptosis, caspase-3 and caspase-7 proteins are activated and able to cleave the caspase 3/7 recognition sequence encoded in the DEVD peptide. Cleavage of the recognition sequence and binding of DNA by the reagent labels the apoptotic cells with a bright, fluorogenic signal. When used together with the appropriate SYTOX dead cell stain, apoptotic cells can be easily discriminated from live and necrotic cells (Figure 6).
Figure 6. Caspase activity detection in Jurkat cells using the CellEvent Caspase-3/7 Green Flow Cytometry Assay Kit on the Attune Flow Cytometer. Jurkat cells (T-cell leukemia, human) were treated with (A) DMSO or (B) 10 µM camptothecin for 3 hours before labeling with the CellEvent Caspase 3/7 Green Flow Cytometry kit. Stained samples were analyzed on the Attune Acoustic Focusing Cytometer equipped with a 488-nm laser, and fluorescence emission was collected using a 530/30 BP filter for CellEvent Caspase 3/7 Green Detection Reagent and a 690/50BP filter for SYTOX AADvanced stain, respectively. Note that the treated cells have a higher percentage of apoptotic cells (panel B) than the basal level of apoptosis seen in the control cells (panel A). A = apoptotic cells, V = viable cells, N = necrotic cells.
End-point caspase assays for flow cytometry
The Vybrant FAM Caspase Assay Kits, also known as the Vybrant FLICA Caspase Apoptosis Assay Kits, are easy to use and do not require the need for special buffers to lyse or permeabilize the cell membranes. Simply add the reagent to your cell culture media, let it incubate 1–4 hours, wash the cells, and analyze (Figure 7).
Figure 7.Detecting active caspases in apoptotic cells using the Vybrant FAM Caspase Assay Kits. Unbound FLICA reagent diffuses out of the cell and is washed away. The green-fluorescent signal is a direct measure of the amount of active caspase that was present at the time the inhibitor was added.
These assays employ the fluorescent inhibitor of caspases (FLICA) methodology. The FLICA reagent, a caspase inhibitor, binds covalently and irreversibly to the reactive cysteines of active caspases. This binding inhibits further enzymatic activity and makes this an end point assay for caspase activity (Figure 8). Vybrant FAM Caspase Assay Kits allow for the accurate detection of active caspases in living cells, are more reliable than annexin V, and simpler to use than TUNEL assays.
Figure 8. Detection of active caspases in living cells by flow cytometry. Jurkat cells were either treated with 10 μM camptothecin for 4 hours at 37ºC and 5% CO2 and stained with the FLICA reagent for caspase-3 and -7 and propidium iodide, both from the Vybrant FAM Caspase-3 and -7 Assay Kit. Samples were analyzed on a flow cytometer with 488 nm excitation using 530 nm bandpass and 670 nm longpass emission filters.
Ordering information
- Mcllwain DR, Berger T, and Mak TW (2013) Cold Spring Harb Perspect Biol 7(4): a026716
- Matsuura K, Canfield K, Feng W, et al (2016) Int Rev Cell Mol Biol 327: 43-87
- Opdenbosch N V and Lamkanfi M. (2029) Immunity 50(6): 1352-1364
- Julien O and Wells JA (2017) Cell Death & Differentiation 24: 1380-1389
- Van Opdenbosch N and Lamkanfi M (2019) Immunity 50(6): 1352-1364
- Sahoo G, Samal D, Khandayataray P, et al (2023) Molecular Neurobiology 60: 5805-5837
- Amstad PA, Yu G, Johnson GL et al. (2001) BioTechniques 31: 608-616
- Smolewski P, Grabarek J, Halicka HD et al. (2002) J Immunol Methods 265:111-121
- Pozarowski P1, Huang X, Halicka DH, et al. (2003) Cytometry A 55:50-60
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For Research Use Only. Not for use in diagnostic procedures.