Immunofluorescence Flow Cytometry

Although special pathology techniques such as immunohistochemistry can prove quite useful in evaluating tissues, flow cytometry is more sensitive and allows analysis of a large number of cells in tissues amenable to single-cell analysis. Flow cytometry provides the most basic measure of the cell cycle in G1, S, and G2 phase fractions, calculated from DNA histogram distributions. Additional information is obtained from Ki-67 staining simultaneously with DNA. BrdU pulse labeling of cells in S phase can also be used effectively to examine cell cycle kinetics in mice, followed by analysis with anti-BrdU antibody. Antibodies to cyclins A, B1, D, and E are analyzed simultaneously with DNA content by flow cytometry; tumors often exhibit dysregulated patterns of expression of cyclins throughout the cell cycle, and this is readily visualized by such analyses. Cell death, which often occurs through the apoptotic process, is just as important a parameter in understanding cellular behavior as is cell proliferation and control. Cell death and apoptosis can be readily visualized by flow cytometry.

DNA content, cell cycle, and cell kinetics measurement are used in the assessment of DNA content (ploidy), proliferative activities of cells and tissues, for observing evidence of cell-cycle checkpoint arrest following exposure to DNA damaging agents, and assessing cell survival and death. A broad spectrum of assays for these purposes is available, ranging from simple univariate DNA content measurements, through dual DNA/RNA staining. The BrdU-Hoechst method is based on continuous exposure to BrdU.17 In addition, BrdU can be used for pulse labeling, and the incorporated BrdU can be detected by using an anti-BrdU antibody, or by UV exposure (BrdU containing DNA undergoes photolysis) followed by labeling of resultant DNA strand breaks by terminal transferase incorporation of FITC-dUTP. The cyclical expression of cyclins (e.g., cyclin A, B1, D, E) is believed to be central to the regulation of the cell cycle. Abnormal expression may be associated with conditions of altered cell proliferation. Although cyclin expression can be quantified by a variety of means, only flow cytometry can do so while identifying the expression as a function of the phase of the cell cycle and heterogeneity in expression in a population. The use of anticyclin antibodies simultaneously with DNA staining has emerged as a major area of current interest in cell cycle regulation.18 Antibodies to cyclins B1 and A are especially easily used. Similarly, cell activation can be quantified with antibodies to proliferation-associated antibodies such as PCNA or Ki-67, and abnormal expression of proteins associated with transformation or immortalization, such as p53, can be quantitated. Susceptibility to apoptosis is thought to be related to oxidative states, as agents that are antioxidants can reduce apoptosis. In addition, induction of apoptosis is a cellular response to DNA damage. Thus, measurements of apoptosis are highly relevant to validating mouse cancer models. Flow cytometry has become a method of choice for quantitation of cell and nuclear features characteristic of apoptosis. Early cellular changes leading to apoptosis can be detected by altered membrane characteristics and light scatter.19 The most characteristic feature of apoptosis is fragmentation of nuclear DNA by endonuclease. Loss of DNA fragments from permeabilized cells can be detected, using flow cytometry, by a reduction in cellular fluorescence leading to a subdiploid "apoptotic peak."20 However, a more recent and, in many cases superior, detection method is based on fixation of cells and end labeling the apoptotic DNA fragments using terminal transferase to incorporate fluorescein labeled dUTP.21

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