Application Note:  Incucyte^® Apoptosis Assays for Kinetic Quantification of Apoptosis

Apoptosis is a complex and dynamic process implicated in several pathologies, including neurodegeneration, autoimmune disease and cancer. A deeper understanding of the mechanisms is vital across a range of therapeutic areas.

The activation of caspase-3 or caspase-7 often 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.

The major drawbacks of many traditional common apoptosis assays is that they yield a single arbitrary user-defined end-point measurement, which can vary considerably for different cell types and for different compound treatment conditions. These assays require multiple wash steps or cell lifting, which may result in the loss of dying cells or lead to a loss in PS asymmetry, In addition, these traditional assays are not amenable to long-term measurements due to increasing background signal over time. 

Live-cell imaging and analysis methods have been developed, however optimized technology and reagents are required for flexible and accurate assessment of cell death. In this application note, we describe the value of Incucyte^® Apoptosis Assays, which encompass no-wash mix-and-read reagents, and integrated image-based analysis tools optimized for kinetic quantification of apoptosis. 

Combining Incucyte^® Caspase-3/7 or Annexin V Dyes with real-time image analysis using an Incucyte^® Live-Cell Analysis System, in physiologically relevant conditions, provides insight into the dynamics and timing of the apoptotic signaling pathway. These assays eliminate the need for determining a single, optimum end-point measurement.

This approach also enables validation of data using the acquired images and assessment of morphological changes associated with cell death. Further insight can be gained through
multiplexing apoptosis readouts with measurements of proliferation, which in combination with Incucyte^® Cell-by-Cell Analysis, has the added benefit of being able to track the cell health of individual subpopulations of cells. 

Together, these assays provide novel and flexible methods, which enable apoptotic pathway analysis for both drug discovery as well as basic cell biology research.

Validated data highlighted in this Application Note include:

• Quantification of Apoptosis
• Pharmacological, Kinetic Analysis of Apoptosis
• Multiplexed Measurements of Proliferation and Apoptosis
• Assessment of Cell Health in Subpopulations of Cells

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