Apoptosis
Apoptosis can be defined as 'gene-directed cellular self-destruction'; it is sometimes referred to as 'programmed cell death' although this is really a phenomenon where cells are programmed to die at a particular point, e.g. during embryonic development, and even here cells may go through an apoptotic pathway. However, apoptosis is certainly a distinct process from other forms of oncosis leading to necrosis.
Necrosis |
Apoptosis |
| Affects groups of cells |
Affects individual cells |
| Non-physiological induction, e.g. virus, poison |
Physiological induction, e.g. lack of signals |
| Phagocytosis of macrophages |
Phagocytosis by macrophages or other cells |
| Inflammatory response |
No inflammatory response |
There are many ways of detecting apoptosis by flow cytometry and this page briefly describes the more common ones in use in this Laboratory. Further details and practical issues such as staining protocols may be obtained by email. Apoptotic cells can be recognised by a characteristic pattern of morphological, biochemical and molecular changes, which may be broadly and chronologically defined as:
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Morphological Changes
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Functional/Biochemical Changes
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- Cell shrinkage
- Cell shape change
- Condensation of cytoplasm
- Nuclear envelope changes
- Nuclear fragmentation
- Loss of cell surface structures
- Apoptotic bodies
- Cell detachment
- Phagocytosis of remains
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- Free calcium ion rise
- bcl2/BAX interaction
- Cell dehydration
- Loss of mitochondrial membrane potential
- Proteolysis
- Phosphatidylserine externalisation
- Lamin B proteolysis
- DNA denaturation
- 50-300kb cleavage
- Intranucleosomal cleavage
- Protein cross-linking
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The methods of detecting apoptosis by flow cytometry in our laboratory are based on the measurement of light scatter, the detection of changes in the plasma membrane, the analysis of cell organelles or the sensitivity of DNA to denaturation. In all methods for the detection of apoptosis, a positive control is useful ie cells in which apoptosis has been induced by, for example, fas induction, treatment with the topoisomerase I inhibitor, camptothecin or treatment with the non-specific protein kinase inhibitor, staurosporine.What to bear in mind about which method to use: - The cell system used (e.g. suspension vs. adherent cells)
- The information being sought
- The technical expertise available
- The lasers available
- The cost, simplicity and number of samples
We should also remember: - Apoptosis in an individual cell is a rapid event but the time taken for a cell to display signs of apoptosis after a stimulus will vary
- Don't assume that signs of apoptosis will be seen in a flow cytometric assay
- Collect the supernatant when using adherent cells
- Use flow cytometry in conjunction with other methods especially morphology but also electron microscopy, time lapse photography and molecular methods (e.g. DNA laddering)
More information about the techniques used here to evaluate apoptosis may be found by clicking the relevant links. There are also several useful websites devoted to apoptosis.
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