One of the most common applications of flow cytometry is measurement of the DNA content of cells. In this way different treatments can be assessed for their effect on cell cycle distribution: for example, treatment with a drug or transfection with a gene of interest. The technique can easily be applied to both mammalian and yeast cells, although due to their much smaller size and DNA content, yeasts show less defined phases of the cell cycle.
There are many different fluorophores that can be used to determine DNA content, each having differing excitation and emission wavelengths. This makes it possible to combine cell cycle analysis with many other different fluorophores. Some dyes, such as DAPI and 7-AAD, are specific to DNA and do not bind to RNA, whereas others, such as propidium iodide, bind to both DNA and RNA and require the removal of RNA (using RNase) before an accurate DNA profile can be obtained.
Most DNA binding dyes are impermeable to live cells (Hoechst 33342 and Vybrant DyeCycle dyes from Invitrogen being the exceptions), which must be permiablised before DNA content can be measured. The most common method of permiablisation is alcohol fixation, which has the added advantage of allowing storage of fixed samples for many weeks before staining. However, alcohol fixation can have a deleterious effect on other cellular characteristics (eg antigen integrity). To overcome these effects, saponin may be used, which allows the DNA dye to enter 'live' cells, making it a more suitable technique in some instances.
When using flow cytometry to assess DNA content, the fluorescence data (FL-A) is acquired with linear amplification and plotted on a single parameter histogram. It is common practice to set the G1 peak at a point on the scale which allows good resolution of the G1, S and G2/M phases while still allowing endoreduplicated/multi-nucleated cells (>4N) to fit on the scale. The quality of cell cycle data can be assessed by looking at the co-efficient of variation (CV) of the G1 peak. The lower the CV the better quality the data but generally a CV of <6% represents acceptable data.
Another benefit of flow cytometry as a means of assessing cell cycle distribution is its ability to exclude doublets from the analysis. Doublets are a problem because a doublet of two G1 cells will have the same DNA content as a single G2/M cell. 'Doublet discrimination' is a process whereby the area (FL-A) of the fluorescence light pulse is plotted against the width (FL-W). Doublets will have greater pulse width than a single cell, as they take longer to pass through the laser beam, and therefore can be excluded from the analysis.
Cell cycle regulated proteins
The expression of many proteins are tightly regulated during cell cycle progression. The detection of these intracellular proteins by flow cytometry, using fluorescenly labelled antibodies in combination with DNA content, enables a more detailed analysis of cell cycle distribution than DNA content alone.
Commonly used markers are phoph-histone H3, to distinguish mitotic cells, cyclin B, which increases during G2 and becomes maximal in mitosis, and cyclin D which increases during the G1 phase of the cell cycle. Using multi-colour flow cytometry it is relatively easy to look at all of these proteins as well as DNA content on an individual cell level.