What is Apoptosis?
Apoptosis is an essential process for normal tissue development and homeostasis by which cells undergo timely programmed cell death. Aberrations in apoptotic signaling are implicated in a range of human pathologies including cancer, autoimmune disease and neurodegeneration. Induction of apoptosis leads, in most cases, to the activation of caspases (cysteinyl aspartate proteinases) and plasma membrane alterations. The activation of caspase-3 or caspase-7 results in the irreversible commitment of the cell to apoptotic death, and is considered a reliable marker for apoptosis. The regulated loss of plasma membrane phosphatidylserine (PS) symmetry is also a classical marker of apoptosis. Dying cells trigger the translocation of the normally inward-facing PS to the cellular surface, allowing for early phagocytic recognition of the dying cell by surrounding phagocytes.
Numerous enzymatic, plate-reader and flow-cytometric assays have been designed to measure caspase-3/7 activation or PS externalization. Most caspase-3/7 assays involve luciferase, colorimetric or fluorometric reagent substrates that incorporate a DEVD (Asp-Glu-Val-Asp) peptide motif which is recognized by the enzyme. Annexin V is a recombinant protein with a high affinity and selectivity for PS residues, allowing it to be used for the detection of apoptosis. Apoptosis assays using Annexin V conjugated to a fluoroprobe have been optimized for detection of PS externalization and are most commonly measured by flow-cytometry. The major drawbacks of these common apoptosis assays are (1) they yield a single, (arbitrary) user-defined end-point measurement; (2) they require multiple wash steps or cell lifting that may result in the loss of dying cells or lead to a loss in PS asymmetry; and (3) they are not amenable to long-term measurements due to increasing signal background over time.
Application
Introducing the Incucyte® Apoptosis Assays
The Incucyte® Live-Cell Analysis System enables real-time, automated apoptosis assays inside your tissue culture incubator. Measure multiple apoptotic pathways simultaneously and in real time using the mix-and-read Incucyte® Caspase-3/7 and Annexin V Dyes. Correlate apoptotic signals with Incucyte® high definition phase contrast images to provide additional biological insight and morphological validation of apoptotic cell death (e.g. cell shrinkage, membrane blebbing, nuclear condensation).
- The Incucyte® Caspase-3/7 Dyes are inert, non-fluorescent (DEVD) substrates that freely cross the cell membrane where they can be cleaved by activated caspase-3/7 to release either a green or red DNA-binding fluorescent label. Apoptotic cells are identified by the appearance of fluorescently-labeled nuclei.
- The Incucyte® Annexin V Dyes are labeled with exceptionally bright and photostable CF dyes that emit a green, red, orange, or Near-IR fluorescent signal upon binding to exposed PS in apoptotic cells.
Measure apoptosis in tumor, immune or neuronal cultures using Incucyte apoptosis assays. Real-time detection of apoptosis in Incucyte® Nuclight Green labeled MDA-MB-231 human breast adenocarcinoma cells treated with TNFα and cycloheximide (left), and in Jurkat T lymphocyte cells treated with camptothecin (right). Apoptotic cells are labeled red using the mix-and-read Incucyte Caspase-3/7 Red Dye or Annexin V Red Dye respectively. Apoptotic cells are quantified in real time using the Incucyte Live-Cell Analysis System.
Incucyte® Apoptosis Assay Concept
- Detect apoptosis in real-time by adding the mix-and-read Incucyte Caspase-3/7 and Annexin V Dyes to your cultures
- Automatically quantify apoptotic cells using intuitive Incucyte® image analysis tools
- Multiplex apoptosis readouts with measurements of proliferation or cytotoxicity by combining with Incucyte® Nuclight nuclear labeling reagents or Incucyte® Cytotox Dyes
Key Advantages
Key Advantages of the Incucyte® Apoptosis Assays
- Visualize and Quantify Apoptosis Using Time-Lapse Imaging - Observe apoptosis over time and quantify apoptotic cells using intuitive Incucyte® image analysis tools. Visualize morphological changes and validate treatment effects with images and movies.
- Automatically Analyze the Timecourse of Apoptosis Inside Your Incubator - Determine how and when treatment effects occurred without removing cells from the stable environment of the incubator - ideal for long-term studies (0 to >10 days).
- Simple Mix-and-Read 96/384-Well Protocols - No Washing, No Fixing, No Lifting - Plate cells, add your treatments along with the Incucyte Caspase-3/7 or Incucyte Annexin V Dyes and read kinetically in the Incucyte Live-Cell Analysis System. Read up to 6 x 384-well plates at once for medium/high-throughput screening.
- Detect and Confirm Apoptosis Through Two Different Pathways - Multiplex Incucyte® Caspase-3/7 and Annexin V Dyes to verify apoptosis as a mechanism of cell death.
- Multiplex With Proliferation and Cytotoxicity Measurements - Combine the Incucyte Apoptosis Assays with Nuclight Reagents or Cytotox Dyes for multiplexed measurements of proliferation/cytotoxicity. Readily discriminate between cytotoxic and cytostatic treatment effects.
Visualize and Quantify Apoptosis Using Time-Lapse Imaging
Figure 1. Quantify apoptosis in your choice of cells using Incucyte apoptosis assays and image analysis tools. (Top far-left) Incucyte images of HT-1080 sarcoma cells in the presence of camptothecin (100 µM) and Incucyte Caspase-3/7 Green Dye. Note the characteristic cell shrinkage and membrane blebbing that accompanies the Caspase-3/7 fluorescent green signal. (Bottom far-left) Incucyte image analysis tools enable automated counting of apoptotic tumor cells (pink mask). (Top center-left) Incucyte images of MDA-MB-231 breast adenocarcinoma cells treated with TNFα and cycloheximide in the presence of Caspase-3/7 Red Dye. (Bottom center-left) Incucyte image analysis tools enable automated counting of apoptotic tumor cells (light blue mask). (Top center-right) Incucyte images of Jurkat T lymphocyte cells in the presence of camptothecin (1 µM) and Incucyte® Annexin V Red Dye. (Bottom center-right) Automated image analysis (blue mask) enables direct detection of apoptotic immune cells. (Top right) Incucyte image of primary rat forebrain neurons in co-culture with astrocytes in the presence of glutamate (300 µM) and Incucyte® Annexin V Green Dye. Neurons are selectively labeled with the Incucyte® Neurolight Red Lentivirus. (Bottom right) Analyzed images (blue mask) positively mark apoptotic cells.
Automatically Analyze the Timecourse of Apoptosis Inside Your Incubator
Figure 2. Quantify treatment effects automatically and non-invasively. Incucyte® Apoptosis Assays allow every well of a 96/384 well plate to be imaged and analyzed automatically to provide a microplate readout of cytotoxicity over time (left). Time-courses reveal concentration-dependent treatment effects (center). Transform data into concentration-response curves to compare pharmacology (right).
Simple Mix-and-Read 96/384-Well Protocols - No Washing, No Fixing, No Lifting
Plate cells, add your treatments along with the Incucyte Caspase-3/7 or Incucyte Annexin V Dyes and read kinetically in the Incucyte Live-Cell Analysis System. Read up to 6 x 384-well plates at once for medium/high-throughput screening.
Detect and Confirm Apoptosis Through Two Different Pathways
Figure 4. Confirm apoptotic cell death with two apoptosis readouts. Detect both Caspase-3/7 and Annexin V signals in your cultures. Automatically determine the time courses of apoptotic cell death.
Multiplex With Proliferation and Cytotoxicity Measurements
Figure 5. Multiplex apoptosis measurements with live-cell label free counting and quantification of time-courses and concentration-dependence of cytotoxicity and proliferation. Camptothecin (1 µM) or cycloheximide (1 µM) treated HT-1080 fibrosarcoma cells in the presence of the Incucyte® Annexin V Green Dye to detect apoptotic cells at 24h (masking of dead cells shown). Classification plots to identify green (apoptotic) population (second column), time-course plots (third column) and concentration-response curves (forth column) show difference in effect of a cytotoxic and cytostatic compound.
Frequently Asked Questions
Yes, in principle. The Caspase-3/7 and Annexin V protocols have been optimized using a range of adherent and non-adherent cell lines and should therefore be applicable to any cell type including cancer cells, immune cells and neurons. Please note some cell types may lack expression of Caspase-3 (e.g. MCF-7 cells). For these cells types we would recommend using the Incucyte® Annexin V Reagents rather than Caspase-3/7 Reagent for apoptosis measurements.
Yes. The protocols and data described here show that it is possible to quantify apoptosis in suspension cell lines such as Jurkat human T lymphocyte cells. You may want to coat your plate to ensure your suspension cells stay in the field of view. Coatings such as poly-L-ornithine, poly-D-lysine or Matrigel™ have been shown to work well.
Yes. As long as the fluorophore is compatible with the Incucyte® Live-Cell Analysis System and does not interfere with the fluorescence emitted by the Incucyte® Caspase3/7 or Annexin V Reagents used.
Yes. The protocols and data described here show that it is possible to quantify two markers of apoptosis (e.g. caspase-3/7 activation and phosphatidylserine externalization) in a single well. For this we recommend that you duplex Incucyte® Annexin V Red Reagent (Cat No. 4641) with the green Incucyte® Caspase-3/7 Reagent (Cat No. 4440).
Yes. Duplex the Incucyte® Caspase-3/7 Reagent (Cat No. 4440) with the Incucyte® Red Cytotox Reagent (Cat No. 4633) or combine the opposing red and green versions of the Incucyte® Annexin V Reagent (Cat No. 4641 or 4642) with Incucyte® Cytotox Reagents (Cat No. 4632 or 4633).
Yes. It is possible to duplex the Incucyte® Annexin V Red Reagent (Cat No. 4641) with Propidium Iodide. We recommend quantifying the Propidium Iodide signal using the red channel of the Incucyte® system and setting a spectral unmixing parameter of 80% Red from Green (please refer to the Fluorescent Dye Optimization Technical Note for more information).***
Data has been generated with both the 10x and 20x objectives. The 10x objective offers the benefit of a greater field of view and reduces the number of images required per well. For higher spatial resolution the 20x objective is recommended.
Incucyte® Apoptosis Assays can be performed using any clear sided or black sided 96 or 384-well clear-bottomed micro-titre plate that is supported on the Incucyte® ZOOM system. We have used Corning 96-well plates (Cat # 3904) for the experiments described here.
No. The Incucyte® Annexin V Reagents have been specially formulated for live-cell imaging with the Incucyte® system. Simply mix-and-read. Washing or fixing is not required.
Yes. There is no requirement to use a specialized assay media or buffer when using Incucyte® Annexin V or Caspase-3/7 Reagents with the Incucyte® system. Integrated Incucyte® image analysis tools enable you to minimize any background fluorescence that may arise from the media, reagents or test agents used. Please do note however that binding of Annexin V to externalized phosphatidylserine is Ca2+-dependent, therefore ensure the Ca2+ concentration in the assay media is ≥1 mM.
We recommended counting apoptotic cells when using the Incucyte® Caspase3/7 Reagent for Apoptosis. The Caspase-3/7 Reagent labels the nuclei of apoptotic cells so it is easy to differentiate between the discretely labelled nuclei of neighboring apoptotic cells. The Incucyte® Annexin V Reagents, however, label the entire cell surface. As such, distinguishing neighboring cells can be challenging. When using Annexin V Reagents we recommend quantifying the total fluorescence area as this is a more robust approach.
Yes, measurements of apoptosis can be normalized to label-free Incucyte® cell confluence measurements. Although some treatments may cause significant cell fragmentation or substantial changes in cell phase contrast we have found the Incucyte® phase contrast confluence metric to be an informative estimate of total cell number over the course of the assay.
It is also possible to normalize to the total number of DNA containing objects at the end of the apoptosis assay. We recommend using Vybrant® DyeCycle™ Green Stain (ThermoFisher) for this purpose. The stain can be added directly to the assay wells without aspiration or washing of the apoptosis reagent containing media.
Ordering Information
Ordering Information
The Incucyte Caspase 3/7 and Annexin V Dyes are fully validated for use with the Incucyte Live-Cell Analysis System. In addition, they can be combined with our range of Incucyte Nuclight nuclear labeling reagents, or the Incucyte Cytotox Dyes for multiplexed measurements of proliferation and cytotoxicity alongside apoptosis within the same well.
Product | Qty. | Cat. No. |
|---|---|---|
| Incucyte® Caspase-3/7 Green Dye for Apoptosis | One vial: 20 μL (100-200 tests) | |
| Incucyte® Caspase-3/7 Red Dye for Apoptosis | One vial: 20 μL (100-200 tests) | |
| Incucyte® Caspase-3/7 for Metabolism Dye | One vial: 20 μL (100-200 tests) | |
| Incucyte® Annexin V Green Dye | One vial: 100-200 tests | |
| Incucyte® Annexin V Red Dye | One vial: 100-200 tests | |
One vial: 100-200 tests | ||
One vial: 100-200 tests | ||
One Kit: | ||
Five vials: 5 μL (100 tests) | ||
Five vials: 5 μL (100 tests) | ||
One vial: 100 μL (500-1000 tests) |
