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Functional Analysis
As long as the relevant functional aspect of the cell can be tagged with a detectable marker, flow cytometry can be used to assess functionality. Applications include:
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Calcium Flux measurement.
Calcium is a very important intracellular ion. It plays a vital role in the transduction of signals from the cell membrane to the cell cytoplasm and a change in intracellular calcium levels is a good indication that the cell is responding to a stimulus. Many stimuli can cause mobilisation of calcium either as an influx from the extracellular medium or the release of intracellular stores. By using fluorescent dyes this mobilisation can be observed. The most common method is to use the UV-excited dye Indo-1, which fluoresces at a different wavelength when it is bound to calcium than when it is not. By measuring fluorescence at two different wavelengths and by ratioing the two signals, a flux can be seen. A ratiometric method such as this is useful as it is unaffected by such things as cell size, dye concentration, fluctuations in laser power and so on.
A protocol is available here in PDF or HTML format.
Live cell measurement by Fluorescein Diacetate.
One of the more elegant uses of a flow cytometer is to assess enzyme activity by loading cells with a non-fluorescent substrate which may be acted on by an enzyme to yield a fluorescent product. Fluorescein diacetate will enter live cells and be cleaved by intracellular esterases to yield a fluorescent product (fluorescein) - in this way live cells will fluoresce green and dead cells may also be identified by addition of propidium iodide as in the example below
A protocol for FDA staining is available in PDF or HTML format.
Cell Proliferation by CFSE
CFSE (Carboxyfluorescein Succinimidyl ester) is a lipophilic dye that is taken up by cell membranes. If cells are labelled with this dye and then induced to proliferate, after mitosis each daughter cell will contain half as much dye as the parent and will be half as bright. After each division the level of fluorescence will halve and so measurement of the green fluorescence can be used to monitor the number of divisons. In the example below, human T cells have been labelled and stimulated - we are able to follow divisions here for up to 6 generations. Using a modelling program such as ModFit, we can also determine how many cells belong to each generation.
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