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Apoptosis - Cell Membrane Analysis
In normal cells, phosphatidylserine (PS) residues are found in the inner membrane of the cytoplasmic membrane. During apoptosis, the PS residues are translocated in the membrane and are externalised. In general, though not always, this is an early event in apoptosis and is thought to be a signal to neighbouring cells that a cell is ready to be phagocytosed. Annexin-V is a specific PS-binding protein that can be used to detect apoptotic cells. Annexin-V is available conjugated to a number of different fluorochromes.
In the example below, thymocytes have been treated with dexamethasone to induce apoptosis. Early apoptotic cells are annexin positive but (in this case) PI negative. Because the cells aren't fixed we can exclude dead cells and it is possible to add further markers if the cytometer set-up is appropriate. As with all live cell assays, we have to remember that we are only looking at a snapshot of the cells as they are at time of analysis and generally all apoptotic experiments should be performed over a time course.
Hoechst 33342 is a DNA-binding dye that is able to quantitatively stain the DNA of live cells. However it has also been found that if the concentration of Hoechst is low, the apoptotic cells take up the Hoechst more rapidly. If we also add PI or TO-PRO-3 we can specifically identify the dead cells. This is a rapid and quantitative method but requires the use of a UV laser.
The advantage of using TO-PRO-3 is that cell phenotyping using FITC- and PE-labelled antibodies is also possible. In the example below, thymocytes labelled with CD4-PE and CD8-FITC can be assessed for apoptosis using Hoechst and TO-PRO-3.
Click on the image to view the diagram in full
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A third way of assessing the membrane changes in apoptosis is to use YO-PRO-1 (Molecular Probes). As this fluorochrome emits in the green, it can be combined with propidium iodide to identify dead cells. The rationale here is that cells in early apoptosis are unable to pump out YO-PRO-1 but are still not permeable to other dead cells discriminatory dyes. In this example, Jurkat cells have been induced with staurosporine for 4 hours.
The following protocols are available:- Annexin protocol in PDF or HTML format
- Hoechst/TO-PRO protocol in PDF or HTML format
- YO-PRO-1 protocol in PDF or HTML format
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