Histograms, flow cytometry

Traill, K. N., Bock, G, Winter, U, Hilchenbach, M., Jurgens, G., and Wick, G. (1986) Simple method for comparing large numbers of flow-cytometry histograms exemplified by analysis of the CD4 (T4) antigen and LDL receptor on human peripheral-blood lymphocytes. J Histochem. Cytochem. 34, 1217-1221... 

The ability of some fluorescent dyes to bind DNA quantitatively is exploited in flow cytometry to determine the DNA content of a cell. Dyes such as propidium iodide that bind double-stranded DNA stoichiometrically can be used for the purpose. The intensity of red fluorescence is directly related to the amount of DNA bound by propidium iodide. By comparing the fluorescence intensity of the test specimen and, in turn, its DNA content to the fluorescence intensity of specimens containing normal diploid amounts of DNA, a DNA histogram can be generated. By computing a DNA index, which is the ratio of DNA content of a test specimen to the DNA content of a specimen containing a normal diploid population, information related to the presence of an aneuploid tumor population can be obtained. The DNA index of 1 would imply that the DNA in the test specimen is from a normal diploid population (2N DNA), whereas the DNA index of an aneuploid population will be greater or less than 1. Thus, the DNA index of a tetraploid (4N DNA) would be 2. 

The analysis of cell-cycle progression was one of the earliest applications of flow cytometry (for review, see Darzynkiewicz et al., 2004). In this assay, fluorescence signals from cells stained with DNA-binding fluorochromes are plotted as DNA content histograms that may be analyzed by using histogram deconvolution software to quantify cell-cycle phase distributions (Rabinovitch 1994). Fluorochromes that are useful for this purpose are the plasma membrane-impermeant DNA stains, propidium iodide (PI),... 

The limitations of the application of conventional detergents mentioned above can be circumvented by replacing this approach with cell membrane permeabilization by microwave heating. Improved detection of intracellular antigens can be obtained with microwave heating used in combination with flow cytometry. This approach yields histogram patterns that show clear discrimination between intact cells and cell debris (Fig. 9.5). 

CV The coefficient of variation (CV) is defined as the standard deviation of a series of values divided by the mean of those values (expressed as a percentage). It is used in flow cytometry to describe the width of a histogram peak. Whereas in some proto-... 

Histogram luminescence. Volume 2(11). Single-parameter plot of data. In flow cytometry, the horizontal axis displays the light scatter or fluorescence intensity parameter and the vertical parameter displays the number of events (e.g., cell count). Volume 1(5). 

After incubation started, cell cycle analysis by flow cytometry and cell cycle histograms were recorded for BOA-treated and control plants every 2 h, until 12 or 14 h of incubation with BOA. At least 10,000 nuclei from each sample must be analyzed in the flow cytometer. 

Figure 5.4 Reversibility of cell cycle arrest by apigenin. SW480 cells were cocultured with 80 pM of apigenin for 48 h, washed twice with PBS, and then recultured in fresh media without apigenin for up to 72 h. At the times indicated in the bottom of the panel, histograms of cellular DNA content were obtained by flow cytometry. (From Wang et al.. Molecular Carcinogenesis, 28, 102-110, 2000. With permission.)...

The samples were processed and stained with propidium iodide according to the method described in Ref. [14.8]. Flow cytometry DNA analysis was performed using a FACScan cytometer. The fluorescence intensity of the DNA labelled with propidium iodide was processed using CellQuest software, and the histograms of the DNA content were analysed using ModFit software. For each sample, 20 000 events were acquired. The DI was calculated with reference to the DNA diploid from the same sample. 

Fig. 3. HIV-1 envelope signaling triggers actin rearrangement. Resting CD4T cells were treated with a laboratory-adapted viral strain, HIV-1 4.3 (a), or with a primary viral isolate, HIV-1 g3ug 4e (b), or with gpl 20IIIB (100 pM) (c) for various times, fixed and permeabilized, and then stained with FITC-phalloidin for F-actin and analyzed by flow cytometry. Shown are histograms of F-actin staining.

The result of a flow cytometry experiment is a histogram of cell distribution by the total amount of surface-bound chemokine, with or without an unlabeled competitor Hgand. This assay provides an easy way to quickly screen numerous candidate disulfide-trapped construct combinations uHth-out the complexities of sample purification however, the utUity of this method is limited to chemokines that are detectable on the cell surface by a... 

Data are analyzed by flow cytometry analysis software (BD FACS Diva, BD Biosciences Flowjo, TreeStar). Data presentation using the frequency of positive events is appropriate only in presence of a bimodal distribution of the emitted fluorescence. In this case, there is no particular preference for the use of the histogram or the dot plot to present data. In case of a non-bimodal distribution of the emitted fluorescence, data should be reported as relative MFI (i.e., the ratio between the MFI values of the sample stained with aU the experimental markers and the MFI values of the negative control sample) and the histogram layout should be preferred. Differently from frequency data, this analytical approach provides relative quantitative information of the chemokine receptor expression levels on the surface of each... 

To get DNA histograms, after separating by tripsinization, cells were fixed with 20% ethanol. They were treated with 0.25% ribonuclease for 1 hr followed by the staining of intranuclear DNA with 0.005% propidium iodide. DNA histograms were drawn by flow cytometry (Cytofluorograf 50H, Ortho Co.) followed by cell cycle analysis. ... 

Cell growth was inhibited dose-dependently after exposure to the test solutions with concentrations from 10 ig/ml to 100 pg/ml (Fig. 1). Fig. 2 shows a normal cell surface as a control. Four min exposure to 50 Xg/ml of the drug caused a decrease in cell population of 20% and a partial loss of microvilli (Fig. 3). Flow cytometry did not reveal any abnormal DNA histograms (Fig. 4). 

Joensuu, H. and Kallioniemi, O. P. (1989) Different opinions on classification of DNA histograms produced from paraffin-embedded tissue. Cytometry 10,711-717. van Dam, P. A., Watson, J. V., Lowe, D. G., and Shepherd, J. H. (1992) Flow cytometric measurement of cell components other than DNA virtues, limitations, and applications in gynecologic oncology. Obstet. Gynecol. 79,616-621. 

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