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A Real-Time, Bioluminescent Apoptosis Assay.

Kevin R Kupcho1, Andrew L Niles2

  • 1Promega, Madison, WI, USA.

Methods in Molecular Biology (Clifton, N.J.)
|May 25, 2021
PubMed
Summary

This novel bioluminescent assay tracks phosphatidylserine (PS) exposure in real-time, overcoming timing issues in apoptosis studies. It offers kinetic resolution of cell death, from early to late phases, for various inducers.

Keywords:
Annexin VApoptosisBioluminescentHomogenousMultiplexedNanoBiT™NecrosisNo-washPhosphatidylserineReal-time

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Area of Science:

  • Cell Biology
  • Biochemistry
  • Assay Development

Background:

  • Traditional apoptosis assays face timing challenges with unknown inducer potentials.
  • Monitoring phosphatidylserine (PS) translocation is crucial for detecting early apoptosis.
  • Existing methods often require multiple steps and lack real-time kinetic data.

Purpose of the Study:

  • To present a real-time, bioluminescent assay for continuous apoptosis monitoring.
  • To overcome the 'timing conundrum' associated with traditional apoptosis detection.
  • To provide kinetic resolution of dose- and agent-dependent apoptotic responses.

Main Methods:

  • Utilized a homogenous, no-wash, plate-based assay.
  • Employed annexin V fusion proteins with complementing NanoBiT™ luciferase subunits.
  • Incorporated a time-released luciferase substrate and a fluorescent membrane integrity reagent.

Main Results:

  • The assay continuously reports phosphatidylserine (PS) translocation during apoptosis.
  • Luminescence signal is proportional to PS exposure, indicating early apoptotic events.
  • Fluorescence intensity correlates with secondary necrosis, allowing late-phase detection.

Conclusions:

  • This bioluminescent technique offers exquisite kinetic resolution of apoptosis.
  • The assay circumvents timing issues, enabling precise analysis of cell death kinetics.
  • Compatible with additional reagents for orthogonal cell health measurements.