Applications

Apoptosis

Overview

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 externalisation. 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 optimised for detection of PS externalisation 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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Apoptosis Assays

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 Reagent is an inert, non-fluorescent (DEVD) substrate that freely crosses the cell membrane where it can be cleaved by activated caspase-3/7 to release a DNA-binding green fluorescent label. Apoptotic cells are identified by the appearance of green-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.

apoptosis video 2

apoptosis video 1

Measure apoptosis in tumor, immune or neuronal cultures using IncuCyte apoptosis assays. Real-time detection of apoptosis in A549 carcinoma cells treated with TNFα and cycloheximide (left), and in Jurkat T lymphocyte cells treated with camptothecin (right). Apoptotic cells are labeled green or red using the mix-and-read IncuCyte Caspase-3/7 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

Key Advantages of the IncuCyte Apoptosis Assays

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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

apoptosis reagents

Quantify apoptosis in your choice of cells using IncuCyte apoptosis assays and image analysis tools. (Top left) IncuCyte images of HT-1080 sarcoma cells in the presence of camptothecin (100 µM) and IncuCyte Caspase-3/7 reagent. Note the characteristic cell shrinkage and membrane blebbing that accompanies the Caspase-3/7 fluorescent green signal. (Bottom left) IncuCyte image analysis tools enable automated counting of apoptotic tumor cells (pink mask). (Top center) IncuCyte images of Jurkat T lymphocyte cells in the presence of camptothecin (1 µM) and IncuCyte® Annexin V Red Reagent. (Bottom center) 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 occured 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

0427-B00-Annexin V Reagents for Apoptosis QuickStart

View the IncuCyte Apoptosis Protocol

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
  • Correlate apoptosis with measurements of cell growth – normalize apoptosis values to label-free confluence measurements for a real-time apoptotic index

Confirm apoptotic cell death with two apoptosis readouts – normalize to cell growth for a real-time apoptotic index. Detect both Caspase-3/7 and Annexin V signals in your cultures. Automatically determine the time courses of apoptotic cell death and correlate with label free confluence measurements to provide an estimate of the proportion of apoptotic cells within the population (apoptotic index).


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

Quantify time-courses and the concentration-dependence of apoptosis and proliferation. Effect of cycloheximide on HT1080 cells: (left) Apoptosis (center) Cell Count and (right) Concentration-response curves.

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Ordering Info

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.

Product Product Data Sheet Safety Data Sheet (US) Safety Data Sheet (EU) Qty. Catalog No.
IncuCyte® Caspase-3/7 Reagent for apoptosis -- 20 μL 4440
IncuCyte® Annexin V Red Reagent for apoptosis 100 tests 4641
IncuCyte® Annexin V Green Reagent for apoptosis 100 tests 4642
IncuCyte® Cytotox Red Reagent for counting dead cells -- 5 μL x 5 4632
IncuCyte® Cytotox Green Reagent for counting dead cells -- 5 μL x 5 4633
IncuCyte® NucLight Red BacMam 3.0 Reagent for nuclear labeling -- 1 mL 4621
IncuCyte® NucLight Green BacMam 3.0 Reagent for nuclear labeling -- 1 mL 4622

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