Apoptosis assays.
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.
Oancea
Marcela
M
Department of Cancer Biology, Cleveland Clinic Lerner Research Institute, OH, USA.
Mazumder
Suparna
S
Crosby
Meredith E
ME
Almasan
Alexandru
A
D017304:Annexin A5; D017209:Apoptosis / Q000502:physiology*; D006690:Bisbenzimidazole; D002318:Cardiovascular Diseases / Q000503:physiopathology*; D053148:Caspase 3 / Q000032:analysis; D002458:Cell Fractionation; D002462:Cell Membrane / Q000502:physiology; D003124:Colorimetry; D045304:Cytochromes c / Q000032:analysis; D003584:Cytological Techniques / Q000379:methods*; D053938:DNA Fragmentation; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008928:Mitochondria / Q000378:metabolism; D013778:Tetrazolium Salts; D014343:Trypan Blue; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000032:analysis; D051028:bcl-2-Associated X Protein / Q000032:analysis
D017304:Annexin A5; D013778:Tetrazolium Salts; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D053148:Caspase 3; D014343:Trypan Blue; D006690:Bisbenzimidazole