Apoptosis Assays for Live-Cell Analysis

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.

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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 Reagents. 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 Reagents 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 Reagents are labeled with exceptionally bright and photostable CF dyes that emit a green or red 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 Reagent or Annexin V Red Reagent respectively. Apoptotic cells are quantified in real time using the Incucyte Live-Cell Analysis System.

Incucyte Apoptosis Assay Concept

1. Detect apoptosis in real-time by adding the mix-and-read Incucyte Caspase-3/7 and Annexin V Reagents to your cultures
2. Automatically quantify apoptotic cells using intuitive Incucyte® image analysis tools
3. Multiplex apoptosis readouts with measurements of proliferation or cytotoxicity by combining with Incucyte® NucLight nuclear labeling reagents or Incucyte® Cytotox Reagents

Key Advantages of the Incucyte Apoptosis Assays

• Visualize and quantify apoptosis using time-lapse imaging
• Automatic analysis of the time course of apoptosis inside your incubator
• Simple mix-and-read 96/384-well protocols - no washing, no fixing, no lifting
• Detect and confirm apoptosis through two different pathways
• Multiplex with proliferation and cytotoxicity measurements

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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

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 Apoptosis reagent. 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 Apoptosis reagent. (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 Reagent. (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 Reagent. Neurons are selectively labeled with the Incucyte® NeuroLight Red reagent. (Bottom right) Analysed images (blue mask) positively mark apoptotic cells.

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)

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 IncuCye Caspase-3/7 or IncuCye Annexin V Reagents and read kinetically in the Incucyte 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 Reagents to verify apoptosis as a mechanism of cell death
  
  
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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

• Combine the Incucyte Apoptosis Assays with NucLight or Cytotox Reagents for multiplexed measurements of proliferation/cytotoxicity. Readily discriminate between cytotoxic and cytostatic treatment effects.
• Capture proliferation label-free, using the Incucyte Cell-by-Cell Analysis Software Module, enabling individual cell segmentation and classification based on fluorescence. Calculate the cytotoxic or apoptotic index within the Incucyte Software.

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 HT1080 fibrosarcoma cells in the presence of the Incucyte® Annexin V Green Reagent 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.

Ordering Information

The Incucyte Caspase 3/7 and Annexin V Reagents 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 Reagents for multiplexed measurements of proliferation and cytotoxicity alongside apoptosis within the same well.