Analysis of Cellular DNA Content by Flow and Laser Scanning Cytometry

Abstract

This chapter covers several aspects of methodology of DNA content analysis in individual cells that is most commonly used for assessment of DNA ploidy and for enumeration of cells in particular phases of the cell cycle. Briefly presented are general principles of instrumentation and cell analysis by flow- and laser scanning- cytometry. Described are major methods designed to stain DNA with fluorochromes in live cells, in detergent-permeabilized cells, in cells fixed prior to DNA staining as well as in nuclei of cells isolated from paraffin-embedded tissues. Briefly addressed are approaches to estimate cellular DNA content in conjunction with cellular immunophenotype. Discussed are factors that affect accuracy of DNA content measurement such as: (i) differences in chromatin structure of the analyzed cells that restrict DNA accessibility to fluorochromes, (ii) stoichiometry of interaction between fluorochromes and DNA in chromatin and (iii) chemical mass action law defining dependency of fluorochrome binding to DNA in relation to fluorochrome concentration and number of potential binding sites in a sample. Described also are controls used to ensure accuracy of DNA ploidy determination, the principles in ploidy assessment and possible pitfalls in analysis.

Prognostic significance of S-phase fractions in peritumoral invading zone analyzed by laser scanning cytometry in patients with high-grade glioma: A preliminary study

“…LSC was developed to utilize the sensitivity of fluorescence-based assays and the specificity of on-slide measurements (16)(17)(18). LSC involves a microscope-based cytofluorometer that is able to assess flow and image cytometry.…”

The Use of Flow Cytometric DNA Ploidy Analysis of Liver Biopsies in Liver Cirrhosis and Hepatocellular Carcinoma

“…Interphase is further divided into three sub-phases, G1, S, and G2. In G1, cells integrate environmental and internal signals that are aimed at the "replicate"/"do not replicate" decision (Darzynkiewicz et al, 2010). The S phase is defined by the ability to synthesize genomic DNA.…”

“…The assessment of cellular kinetics of liver biopsies is of great importance for understanding the development of liver disease. Cellular DNA content can be measured by flow cytometry (FCM) with the aim of (1) revealing cell distribution within the major phases (G0/G1, Sphase and G2/M) of the cell cycle, (2) estimating the frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing the DNA-ploidy of the measured cell population (Darzynkiewicz et al, 2010). On the other hand, Argyrophilic nucleolar organizer regions (AgNORs) proteins are a set of argyrophilic nucleolar proteins that accumulate in highly proliferating cells, whereas their expression is very low in nonproliferating cells (Fariña et al, 2011).…”

Raman spectroscopy for DNA quantification in cell nucleus

“…Three major approaches to measure nuclear DNA content exist: these include fluorescence flow cytometry, cytophotometry of Feulgen-DNA cytophotometry, and laser scanning cytometry (1). Flow cytometry relies on detecting the fluorescent signal emitted by a single cell or a nucleus as it traverses the path of a laser beam, with signal intensity being translated into DNA content (2).…”

“…Laser scanning cytometry involves scanning and integrating the total fluorescence of a nucleus. The cell nucleus is known to encompass DNA sequences associated with various protein complexes, thereby accessibility of such DNA regions to dyes is nonuniform and is a factor that limits the accuracy of these methods (1).…”