Incucyte® Apoptosis Assays for Live-Cell Analysis
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. 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 common apoptosis assays are that they:
- Yield a single, (arbitrary) user-defined end-point measurement
- Require multiple wash steps or cell lifting that may result in loss of dying cells or lead to a loss in PS asymmetry
- Are not amenable to long-term measurements due to increasing signal background over time.
The Incucyte® Live-Cell Analysis System enables real-time, automated apoptosis assays that measure multiple apoptotic pathways simultaneously and in real time using the mix-and-read Incucyte® Caspase-3/7 and Annexin V Dyes.
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.
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).
See example data below
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. No washing, fixing or lifting of cells
See example data below
Multiplex Incucyte® Caspase-3/7 and Annexin V Dyes to verify apoptosis as a mechanism of cell death.
See example data below
Combine Incucyte® Apoptosis Assays with Incucyte® Nuclight Reagents or Cytotox Dyes for multiplexed measurements of proliferation/cytotoxicity. Readily discriminate between cytotoxic and cytostatic treatment effects.
See example data below
Example Data for Apoptosis Assays
Visualize, 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 Incucyte® 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 Time-course 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 cell death over time (A). Time-courses reveal concentration-dependent treatment effects (B). Transform data into concentration-response curves to compare pharmacology (B). Validate quantification with images at every time point (C).
Simple Mix-and-Read 96/384-Well Protocols - No Washing, No Fixing, No Lifting
Figure 3. 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.
Solutions for Apoptosis Assays
Incucyte® Apoptosis Assay Overview
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 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).
- 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.
- 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.
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Apoptosis Assays Technical Resources
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Apoptosis Assays Frequently Asked Questions
Yes, in principle. Incucyte® 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 Dyes rather than Incucyte® Caspase-3/7 Dye for apoptosis measurements.
Yes. The protocols and data described here show 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 signal emitted by the Incucyte® Caspase3/7 or Annexin V Dyes 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 example, for this we might recommend that you duplex Incucyte® Annexin V Red Dye (Cat No. 4641) with the green Incucyte® Caspase-3/7 Dye (Cat No. 4440).
Yes. You can multiplex the Incucyte® Caspase-3/7 Dye or Incucyte® Annexin Dyes with the Incucyte® Cytotox Dyes of a different color to evaluate other cell death readouts.
Yes. It is possible to duplex the Incucyte® Annexin V Red Dye (Cat No. 4641) with Propidium Iodide. We recommend quantifying the Propidium Iodide signal using the red channel of the Incucyte® Live-Cell Analysis 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 shown has been generated with both the 10X and 20X objectives, but the assay is also compatible with the 4X objective. 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-titer plate that is supported on the Incucyte® Live-Cell Analysis System. We have used Corning 96-well plates (Cat # 3595) for the experiments described here.
No. The Incucyte® Caspase 3/7 and Annexin V Dyes 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 Dyes with the Incucyte® Live-Cell Analysis 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 Dye. The Caspase-3/7 Dye stains the nuclei of apoptotic cells so it is easy to differentiate between the discretely labelled nuclei of neighboring apoptotic cells. The Incucyte® Annexin V Dyes, however, label the entire cell surface. As such, distinguishing neighboring cells can be challenging. When using Annexin V Dyes, 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.