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Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
Published on: April 24, 2011
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A Nondestructive, Real-Time Annexin V Apoptosis Assay
Andrew L Niles1, Kevin R Kupcho2
1Promega, Madison, WI, USA. andrew.niles@promega.com.
Methods in Molecular Biology (Clifton, N.J.)
|September 10, 2022
Summary
This new annexin V apoptosis assay offers real-time, nondestructive detection without flow cytometry. It enables precise kinetic analysis of cell death, making apoptosis studies more accessible.
Area of Science:
- Cell Biology
- Biochemistry
Background:
- Apoptosis detection is crucial for understanding cell death.
- Existing methods like flow cytometry are often complex and time-consuming.
- Annexin V is a widely used biomarker for early apoptosis.
Purpose of the Study:
- To describe a simple, real-time, and nondestructive method for detecting apoptosis using annexin V.
- To provide an accessible alternative to flow cytometry-based apoptosis assays.
- To enable kinetic characterization of apoptotic responses.
Main Methods:
- Utilizes optimized annexin V fusion proteins with NanoBiT™ luciferase subunits.
- Employs a time-released luciferase substrate and a fluorescent membrane integrity reagent.
- Designed for standard multimodal plate readers, requiring no wash steps or flow cytometry.
Main Results:
- The assay provides real-time monitoring of phosphatidylserine (PS) translocation during apoptosis.
- Luminescence signal is proportional to the number of cells with PS exposure.
- Fluorescence intensity correlates with secondary necrosis, allowing for temporal resolution of cell death.
Conclusions:
- This homogenous, no-wash assay is accessible to various laboratories.
- It allows for detailed kinetic and potency determinations of apoptotic responses.
- The method facilitates a comprehensive understanding of dose- and agent-dependent cell death.
