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Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
07:34

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

Published on: April 24, 2011

Expression, purification and use of recombinant annexin V for the detection of apoptotic cells

Susan E Logue1, Mohamed Elgendy, Seamus J Martin

  • 1Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, Ireland.

Nature Protocols
|September 5, 2009
PubMed

Insights

This study details a rapid protocol for producing and labeling recombinant annexin V, a key protein for detecting programmed cell death (apoptosis). The method enables efficient analysis of apoptotic cells using flow cytometry or fluorescence microscopy.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Background:

  • Apoptosis, or programmed cell death, is crucial for development and tissue health.
  • Detecting apoptosis involves identifying externalized phosphatidylserine on cell membranes.
  • Annexin V, labeled with fluorescent markers, is a standard tool for this detection.

Purpose of the Study:

  • To provide a streamlined protocol for producing and labeling recombinant annexin V.
  • To enable researchers to generate sufficient quantities of annexin V for experiments.
  • To facilitate the assessment of annexin V binding to cells.

Main Methods:

  • Bacterial expression and purification of recombinant annexin V.
  • Fluorescein isothiocyanate (FITC) labeling of purified annexin V.
  • Assessment of annexin V binding via flow cytometry and fluorescence microscopy.

Main Results:

  • Milligram quantities of recombinant annexin V can be produced within 3 days.
  • The protocol yields functional annexin V suitable for labeling.
  • Methods for analyzing annexin V binding to cell populations are established.

Conclusions:

  • This protocol offers an efficient method for preparing FITC-labeled annexin V.
  • The described techniques support reliable detection and analysis of apoptotic cells.
  • The study facilitates research into programmed cell death mechanisms.

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