Flow Cytometry Laboratory

Apoptosis



Apoptosis can be defined as "gene-directed cellular self-destruction" or programmed cell death. Apoptotic cells can be recognised by a characteristic pattern of morphological, biochemical and molecular changes. These changes can be broadly assigned to three stages:

  • Early

  • Intermediate

  • Late

    There are many ways of detecting apoptosis by flow cytometry and the methods used in our Lab are based on the measurement of DNA content, altered membrane permeability or the detection of endonucleolysis as characterised by DNA strand breaks. It is also possible to detect apoptosis by looking at the light scatter characteristics of cells, or by thier permeability to DNA dyes, although both of these methods are relatively non-specific. At the end of the page is a summary of the methods available, their advantages and disadvantages.



  • Propidium Iodide staining

  • Hoechst and Propidium Iodide staining

  • Strand Break Labelling

  • Annexin V Staining


    Top Propidium Iodide Staining


    Cells are fixed in cold 70% ethanol and then washed twice in phosphate-citrate buffer (192 parts of 0.2M Na2HPO4, 8 parts of 0.1M citric acid).

    This has the effect of extracting the low molecular weight DNA from the apototic cells and so these cells appear to the left of the normal G1 peak. This example is of HL60 cells that have been treated with dexamethasone. This is a quick, easy but not very specific method of detecting apoptosis, although it can give valuable clues to what exactly is happening in a population of cells, and is especially useful over a time course. However apoptosis of cells in late S phase or from G2 may be missed.



    Top Hoechst and Propidium Iodide Staining


    This method relies on the differences in the permeability of the cell membranes of live, dead and apoptotic cells to two DNA dyes - propidium iodide and Hoechst 33342. Dead cells are permeable to both dyes, live cells are able to pump both out and apoptotic cells are able to exclude propidium but not Hoechst. The method is time dependent (the changes are only obvious in the first few minutes after staining), needs a UV laser and is only applicable to certain cell types (thymocytes are excellent). The protocol can be seen here and this is an example of the profile obtained. These are thymocytes that have been treated with dexamethasone.



    Top Strand Break Labelling


    This method uses an enzyme (Terminal deoxynucleotidyl transferase - TdT) to add biotinylated or digoxygenin-labelled nucleotides to the strand breaks found in the DNA of apoptotic cells. They can then be detected by a fluorochrome-labelled streptavidin conjugate or a fluorochrome-labelled anti-digoxygenin antibody. This example is of HL60 cells. Because it is possible to combine this staining with propidium iodide, it is possible to obtain a cell cycle profile and be able to determine from which phase of the cell cycle the cells are beginning to produce strand breaks.



    Top Annexin V Staining


    One of the membrane changes in the early / intermediate stages of apoptosis is the translocation of phosphatidylserine (PS) from the inner of the cell membrane to the outside. It is possible to detect PS by using FITC-labelled Annexin V, which is a Ca2+ dependent phospholipid-binding protein. By combining staining of Annexin-FITC with propidium iodide, we can obtain a similar profile to the Hoechst / PI method, where live cells are negative for both dyes, dead cells are positive for both, while apoptotic cells are positive only for Annexin-FITC as in this example of HL60 cells, which have been treated with camptothecin. The advantage of this method is that it can be used on a benchtop analytical flow cytometer.

    Top Summary of methods available




    METHOD ADVANTAGES DISADVANTAGES
    Light Scatter Easy Suspension cells only
    (FSC v SSC) Semi-quantitative Debris can be a problem
    Phenotyping possible Subjective
    Permeability to DNA dyes Easy Only late apoptosis
    (PI, 7 AAD) . Also detects necrosis
    Membrane changes Quantitative Intermediate apoptosis mainly
    (Annexin V) Easy Phenotyping difficult
    . . More applicable to suspension cells
    Differential permeability Quantitative Intermediate apoptosis mainly
    (Hoechst / PI, LDS) Possible phenotyping May need UV laser
    Hypodiploidy Semi-quantitative Intermediate and late apoptosis
    (Fix then PI) All cell types No phenotyping
    TUNEL All cell types Technically demanding
    Quantitative Intermediate and late apoptosis
    . . Phenotyping possible.




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