Cell Cycle Analysis
The measurement of the DNA content of cells was one of the first major applications of flow cytometry and is still one of the biggest applications in this Laboratory today. The DNA content of the cell can provide a great deal of information about the cell cycle, and consequently the effect on the cell cycle of added stimuli, e.g. transfected genes or drug treatment. We can also combine the measurement of DNA content with the quantitation of an antigen, so that we can assess its expression during the cell cycle, or alternatively look at the DNA profile of a subset of cells defined by our antigen.
How do we define a good DNA peak?
Ideally all cells in the G1 phase will take up the same amount of dye and will all fluoresce in a single channel - but flow cytometers are (unfortunately) not that accurate and there will be minor conformational variations in the DNA leading slightly different amounts of dye being taken up - we quantitate this by using the coefficient of variation (CV) of the G1 peak. The lower the CV the better we can assess DNA changes especially aneuploidy. We have to bear in mind that we are measuring fluorescence from a DNA-binding fluorochrome and inferring that this is the same as DNA content.
However, using a DNA binding dye such as propidium iodide we can get the following profile:
There are a number of dyes that can be used. In general, for quantitative DNA analysis, cells are fixed in ethanol. After this we can use a number of DNA-binding dyes, e.g.:
- Hoeschst 33342
- DAPI
- Mithramicin
- Propidium Iodide
- 7 Aminoactonomycin D
- DRAQ5
- TO-PRO-3
Many of the dyes used to quantitatively stain DNA may also be used to separate live from dead cells in unfixed samples. More information on this can be obtained here.
More about the specific uses of cell cycle analysis in this Laboratory can be found by clicking the appropriate links above.
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