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Identifying the Stages of Apoptosis

  
  
  
  
  

Apoptosis represents a highly regulated multibiochemical event. This pathway plays a key role in cellular development, like Hematopoiesis, and differs from necrosis in that apoptosis is controlled cell death.

Classification of the different types of cell death involves:

  • Definition of the cellular structure
  • Structural changes
  • Membrane alteration
  • Cellular processes changes
  • Nuclear degradation

Apoptosis can be seen as a stage-dependent process from its induction to early, intermediate and late-stage apoptotic events. Methods and reagents have been developed to identify the various apoptotic stages, and to clearly distinguish them from necrotic processes.

Early Stage Apoptosis Identification

Early stage apoptosis is represented by changes to, and ultimate loss of, the mitochondrial membrane potential. Mitochrondrial membrane potential is detected by JC-1, a dye that selectively enters the mitochondria.  In functional mitochondria, JC-1 forms aggregates which show a fluorescent emission at ~590 nm. With reduced membrane potential the dye forms monomers where the emission is shifted to ~530nm. This shift is used to measure mitochondrial membrane potential, and the entry of the cell into early stage apoptosis.

Early/Mid Stage Apoptosis Identification

Another key apoptotic process is the translocation of phosphatidylserin (PS), a protein located in the cellular membrane. PS in live, intact cells is cytosolic but translocates to the extracellular portion of the membrane in early/mid apoptosis. Annexin V can be used for PS detection where cell impermeable dyes, such as Propidium Iodide or 7-AAD, need to be used as a counterstain to differentiate early apoptosis from late apoptosis or necrosis.

Annexin V Apoptosis Detection

Late Stage Apoptosis Identification

You can identify late stage apoptosis by looking at the defragmentation of DNA. Apo-Direct assay utilizes a method to detect the multitude of 3'-hydroxyl ends created during late stage apoptosis. Measurement is provided by a two color stain using fluorescein-deoxyuridine triphosphate (FITC-dUTP) for DNA Break detection and Propidium Iodide for total DNA counterstain.

And these are just a few ways to identify apoptosis….

Additional ways of Apoptosis/Necrosis detection are looking at metabolic changes, membrane changes, Caspases, Phosphoproteins, cellular calcium levels and many more.

Comments

once initiated, is apoptosis reversible? 
 
Can cells road block in the early stage and remain locked in that state? 
 
Can JC-1 be employed as a marker in all stages? 
 
 
 
cheers, 
 
ian
Posted @ Wednesday, October 26, 2011 5:55 PM by ian beckman
The apoptosis process as a whole is not reversible, but there is discussion that some of the individual processes might. When particular apoptosis processes are initiated, cellular control and repair can start and, if successful, the initiated processes may stop.  
There is also speculation that PS translocation is reversible, but there is no conclusion yet and no publications we are aware of discussing this. 
 
JC-1 has been described as one of the most sensitive markers for mitochondrial membrane potential changes and the colorometric change is quite sensitive. But as apoptosis and changes in the mitochondria continue over time, the fluorescence signal is lost.
Posted @ Tuesday, November 01, 2011 7:53 PM by Wendy Parenteau
Mine is not a comment but a question. I read in my Haematology textbook that apoptosis has pro-apoptotic and anti-apoptotic factors in gene form. I want a table containing those factors depending on how they are classified. Please, I need my response ASAP! Thankyou.
Posted @ Wednesday, August 22, 2012 4:44 AM by Tino
I'm afraid we don't have the specific information you are requesting, but we can refer you to the following papers for more information: 
 
 
 
Apoptosis: A review of pro-apoptotic and anti-apoptotic pathways and dysregulation in disease. 
 
Mauria A. O'Brien DVM, Rebecca Kirby DVM, DACVIM, DACVECC 
 
Journal of Veterinary Emergency and Critical Care. Volume 18, Issue 6, pages 572–585, December 2008 
 
 
 
Anti-apoptotic versus pro-apoptotic signal transduction: checkpoints and stop signs along the road to death. 
 
Jarpe MB, Widmann C, Knall C, Schlesinger TK, Gibson S, Yujiri T, Fanger GR, Gelfand EW, Johnson GL. 
 
Oncogene. 1998 Sep 17;17(11 Reviews):1475-82. 
 
<a>http://www.ncbi.nlm.nih.gov/pubmed/9779994 
 
Posted @ Wednesday, August 22, 2012 12:52 PM by Wendy Parenteau
Affraid i have a question too.  
Im looking for the best way to track stages of apoptosis in dopaminergic neurons. Would you happen to have information about this or perhaps kniw where i can find it.
Posted @ Sunday, October 13, 2013 4:12 PM by Coen
Methods and reagents have been developed to identify the various apoptotic stages, to clearly distinguish them from necrotic processes.  
 
The following chart provides a list of products available and is organized according to stage of apoptosis. Check it out at Apoptosis Markers Chart
 
Posted on behalf of eBioscience's Technical Support. For more information, contact them directly at tech@eBioscience.com.
Posted @ Tuesday, October 15, 2013 11:08 AM by Diane Gaige
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