Cell cycle analysis
Cell cycle analysis by DNA content measurement is a method that most frequently employs flow cytometry to distinguish cells in different phases of the cell cycle. Before analysis, the cells are usually permeabilised and treated with a fluorescent dye that stains DNA quantitatively, such as propidium iodide or 4,6-diamidino-2-phenylindole. The fluorescence intensity of the stained cells correlates with the amount of DNA they contain. As the DNA content doubles during the S phase, the DNA content of cells in the G0 phase and G1 phase, in the S phase, and in the G2 phase and M phase identifies the cell cycle phase position in the major phases of the cell cycle. The cellular DNA content of individual cells is often plotted as their frequency histogram to provide information about relative frequency of cells in the major phases of the cell cycle.
Cell cycle anomalies revealed on the DNA content frequency histogram are often observed after different types of cell damage, for example such DNA damage that interrupts the cell cycle progression at certain checkpoints. Such an arrest of the cell cycle progression can lead either to an effective DNA repair, which may prevent transformation of normal into a cancer cell, or to cell death, often by the mode of apoptosis. An arrest of cells in G0 or G1 is often seen as a result of lack of nutrients, for example after serum deprivation.
Cell cycle analysis was first described in 1969 at Los Alamos Scientific Laboratory by a group from the University of California using the Feulgen staining technique. The first protocol for cell cycle analysis using propidium iodide staining was presented in 1975 by Awtar Krishan from Harvard Medical School and is still widely cited today.
Multiparameter analysis of the cell cycle includes, in addition to measurement of cellular DNA content, other cell cycle related constituents/features. The concurrent measurement of cellular DNA and RNA content, or DNA susceptibility to denaturation at low pH using the metachromatic dye acridine orange, reveals the G1Q, G1A, and G1B cell cycle compartments and also makes it possible to discriminate between S, G2 and mitotic cells. The cells in G1Q are quiescent, temporarily withdrawn from the cell cycle, the G1A are in the growth phase while G1B are the cells just prior entering S, with their growth similar to that of the cells initiating DNA replication. Similar cell cycle compartments are also recognized by multiparameter analysis that includes measurement of expression of cyclin D1, cyclin E, cyclin A and cyclin B1, each in relation to DNA content Concurrent measurement of DNA content and of incorporation of DNA precursor 5-bromo-2'-deoxyuridine by flow cytometry is an especially useful assay, that has been widely used in analysis of the cell cycle in vitro and in vivo. However, the incorporation of 5-ethynyl-2'-deoxyuridine, the precursor whose detection offers certain advantages over BrdU, has now become the preferred methodology do detect DNA replicating cells.
Experimental procedure
Unless staining is performed using Hoechst 33342, the first step in preparing cells for cell cycle analysis is permeabilisation of the cells' plasma membranes. This is usually done by incubating them in a buffer solution containing a mild detergent such as Triton X-100 or NP-40, or by fixating them in ethanol. Most fluorescent DNA dyes are not plasma membrane permeant, that is, unable to pass through an intact cell membrane. Permeabilisation is therefore crucial for the success of the next step, the staining of the cells.Prior to the cells are often treated with RNase A to remove RNAs. This is important because
certain dyes that stain DNA will also stain RNA, thus creating artefacts that would distort the results. An exception is the metachromatic fluorochrome acridine orange, which under the specific staining protocol can differentially stain both, RNA and DNA, or in another protocol, after removal of RNA and partial DNA denaturation, to differentially stain double-stranded DNA versus single-stranded DNA . Aside from propidium iodide and acridine orange, quantifiable dyes that are frequently used include DRAQ5, 7-Aminoactinomycin D, DAPI and Hoechst 33342.