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Related Experiment Video

Updated: Aug 29, 2025

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
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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
PubMed
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.

Keywords:
Add-mix-measureAnnexin VApoptosisBioluminescentHomogenousMultiplexedNanoBiT™NecrosisNo-washPhosphatidylserineReal time

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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.