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.
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)
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.
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.
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).
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).
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.
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 Green Reagent for apoptosis||--||20 μL||4440|
|IncuCyte® Caspase-3/7 Red Reagent for apoptosis||--||20 μL||4704|
|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|