DNA Content Measurement for DNA Ploidy unit 75 and Cell Cycle Analysis

In flow cytometry, analysis of DNA ploidy (DNA index or DI) and/or discrimination of cells in G0/G1 versus S versus G2/M phases of the cell cycle is generally done by measuring cellular DNA content alone. Indeed, univariate DNA content analysis is an established clinical assay in oncology and is also widely used for research in cell and molecular biology (see unit 7.1 for an overview of nucleic acid analysis). A large number of DNA fluorochromes can be used for this purpose, and the binding characteristics and spectral properties of most nucleic acid probes are described in unit 4.2. A great variety of techniques for measuring DNA utilizing these fluorochromes have been developed since the mid 1970s (Darzynkiewicz et al., 1994). The techniques differ primarily in the mode of cell permeabilization (detergent versus prefixation with alcohols) and composition of the stain solution. The most commonly used procedures are described in this unit.

Relatively simple and universally applicable methods for staining ethanol-fixed cells are presented in Basic Protocol 1 and Alternate Protocol 1. Because cells may be stored in fixative for extended periods and may be transported while in the fixative, this method allows one to prepare and collect cells independently of the timing of their analysis. The methods presented utilize the two most widely used DNA fluorochromes, propidium iodide (PI; see Basic Protocol 1) and 4',6-diamidino-2-phenylindole (DAPI; see Alternate Protocol 1).

The second set of methods presented (see Basic Protocol 2 and Alternate Protocol 2) utilize detergents and/or hypotonic solutions to permeabilize cells; these methods generally provide more accurate estimates of DNA content compared to measurement of fixed cells. The approach presented in Basic Protocol 2 combines detergent treatment with use of proteolytic enzymes; it is widely used for clinical material, especially for DNA analysis in samples of solid tumors. Alternate Protocol 2 is a simpler method designed for uniform populations (e.g., tissue culture cells).

A third approach is required for DNA content measurements in live cells (see Basic Protocol 3). The primary application of this method is for cell sorting, where cells selected on the basis of differences in DNA content can be subcultured for the purpose of analyzing their growth characteristics, testing their sensitivity to drugs, cloning, or expanding their number. On the other hand, archival samples of paraffin-embedded tissues can be analyzed by flow cytometry following nuclear isolation (see Basic Protocol 4). This methodology is widely used in retrospective studies probing the prognostic value of DNA content in tumors.

Univariate DNA content measurement can also discriminate apoptotic cells, which are characterized by fractional DNA content due to DNA degradation by the apoptosis-asso-ciated endonuclease(s) (Wyllie, 1992; Darzynkiewicz et al., 1997). Apoptotic cells can therefore be identified within a population as the cells that evidence fractional DNA content following extraction of the degraded DNA and subsequent cell staining with PI or DAPI (see Basic Protocol 5). This approach is combined with analysis of DNA degradation by gel electrophoresis (see Support Protocol). The characteristic pattern of DNA degradation, preferentially producing internucleosomal DNA sections and generating so-called DNA laddering on the gels, is considered to be a hallmark of apoptosis. An alternative approach to detecting apoptotic cells by simultaneously measuring DNA content and DNA strand breaks, in which formaldehyde-fixed cells are enzymatically labeled with 5-bromodeoxyuridine triphosphate (BrdUTP) and then costained with Nucleic Acid

Analysis

FITC-conjugated antibody to BrdU (to detect DNA strand breaks) and with PI (to analyze DNA content), is presented in unit 7.4.

Discrimination of cells in particular phases of the cell cycle on the basis of differences in DNA content is helped by computer analysis. The software used to deconvolute the histograms often allows one to measure the cell cycle distribution of both diploid normal cells (host infiltrating and stromal cells) and aneuploid cell populations in aneuploid tumors.

The protocols presented in this unit can easily be modified for use of other DNA fluorochromes. For example, PI, which is used in Basic Protocols 1, 2, and 5, can be replaced by DAPI (e.g., see Alternate Protocols 1 and 2; see Basic Protocol 4), which then is applied at lower concentration and without the need to incubate cells with RNase.

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