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Apoptosis - Light Scatter
As cells die or become apoptotic the refractive index of the internal cytoplasm becomes more similar to that of the extracellular medium - this manifests itself as a reduction in forward scatter signal. At the same time, intracellular changes and invagination of the cytoplasmic membrane lead to an increase in side (or orthogonal or 90°) scatter. If we add a dead cell discriminatory dye we can identify cells that have become permeable. In this way we can get low level resolution of dead and apoptotic cells. A number of dead cell dyes are available for use and the one used will depend on any other fluorochromes that are being measured. Some examples are: - Sytox Green (488nm excitation; green fluorescence emission)
- Propidium Iodide (488nm excitation; orange/red fluorescence emission)
- 7-Aminoactinomycin-D (7-AAD) (488nm excitation; red fluorescence emission)
- TO-PRO-3 (633nm excitation; red fluorescence emission)
In the following example, propidium iodide and TO-PRO-3 have been used to identify dead or late apoptotic cells. Early apoptotic cells are seen as a population showing low dye fluorescence and reduced forward scatter.
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