Apoptosis Assays

  • Marcela Oancea
  • Suparna Mazumder
  • Meredith E. Crosby
  • Alexandru Almasan
Part of the Methods in Molecular Medicine book series (MIMM, volume 129)

Abstract

A large number of methods devoted to the identification of apoptotic cells and the analysis of the morphological, biochemical, and molecular changes that take place during this universal biological process have been developed. Apoptotic cells are recognized on the basis of their reduced DNA content and morphological changes that include nuclear condensation and which can be detected by flow cytometry (sub-G1 DNA content), Trypan Blue, or Hoechst staining. Changes in plasma membrane composition and function are detected by the appearance of phosphatidylserine on the plasma membrane, which reacts with Annexin V-fluorochrome conjugates. Combined with propidium iodide (PI) staining, this method can distinguish between the early and late apoptotic events. The best-recognized biochemical hallmarks of apoptosis are the activation of cysteine proteases (caspases), condensation of chromatin, and fragmentation of genomic DNA into nucleosomal fragments. Recognized by a variety of assays, activated caspases cleave many cellular proteins and the resulting fragments may serve as apoptosis markers. Finally, the mitochondria and the Bcl-2 family proteins play an important role in this process that can be recognized by translocation of apoptogenic factors, such as Bax and cytochrome c, in and out of mitochondria.

Key Words

Apoptosis Bcl-2 family caspase Annexin V binding microscopy flow cytometry methods review 

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Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Marcela Oancea
    • 1
    • 2
  • Suparna Mazumder
    • 1
  • Meredith E. Crosby
    • 3
    • 4
  • Alexandru Almasan
    • 3
    • 5
  1. 1.Department of Cancer BiologyCleveland Clinic Lerner Research InstituteCleveland
  2. 2.Department of ChemistryCleveland State UniversityCleveland
  3. 3.Department of Cancer BiologyLerner Research InstituteCleveland
  4. 4.Department of Environmental Health ScienceCase Western Reserve UniversityCleveland
  5. 5.Department of Radiation OncologyThe Cleveland Clinic FoundationCleveland

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