Flow cytometry cell cycle analysis

One of the first applications developed for flow cytometry was cell cycle analysis.2 There are numerous intercalating fluorescent DNA and RNA staining reagents that can be used to determine the amount of DNA in cells, an indicator of cell cycle stage and progression, as demonstrated in Figure 7.3. Nucleic acid dyes may be selective for DNA... 

N5. Noronha, A., and Richman, D. R, Simultaneous cell surface phenotype and cell cycle analysis of lymphocytes by flow cytometry. J. Histochem. Cytochem. 32, 821-829 (1984). 

Fig. 8.4. Reprinted with permission of John Wiley Sons, Inc. 1990 from Gray JW, et al. (1990). Quantitative cell-cycle analysis. Melamed MR, et al. (eds). Flow Cytometry and Sorting. New York Wiley-Liss, pp 445-467. The work was performed at the University of California Lawrence Livermore National Laboratory under the auspices of the U.S. Department of Energy.

Fig. 8.7. Reprinted (A,D) from Dean PN (1987). Data analysis in cell kinetics. Gray JW and Darzynkiewicz Z (eds). Techniques in Cell Cycle Analysis. Clifton, NJ Humana Press, pp 207-253 and (B,C) from Dean PN (1985). Methods of data analysis in flow cytometry. Van Dilla MA, et al. (eds). Flow Cytometry Instrumentation and Data Analysis. London Academic Press, pp 195-221.

Furthermore, cell cycle analysis by flow cytometry can be improved by using other complementary techniques that provide additional information. This is the case of the mitotic index that informs us about the number of cells undergoing mitosis during the experiment. This measurement was also recorded in our investigation, and the results indicated its excellent complementary information. 

Comparative cell cycle analysis of 6 h hydroxyurea-blocked root cells from 1 mM BOA-treated and control lettuce meristems. Seedlings were exposed to treatment after HU release, and then nuclear suspensions were prepared with 40 meristems per treatment and analyzed by flow cytometry every 2 h. (From Coba de la Pena, T. and Sanchez-Moreiras, A. 2001, Handbook of Plant Ecophysiology Techniques, Kluwer Academica Publishers, Dordrecht, the Nederlands, pp. 65-68. With permission). 

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. 

Belloc, F, Dumain, R, Boisseau, M. R., Jalloustre, C., Reiffers, J., Bernard, P. and Lacombe, R (1994). A flow cytometric method using Hoechst 33342 and pro-pidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells. Cytometry 17, 59-65. 

M. Cell cycle analysis in flow cytometry Use of BrdU labelling and side scatter for the detection of the different 32. cell cycle phases. Cytotechnology 1991, 5 (Suppl. 1),... 

SMOKELESS TOBACCO AND APOPTOSIS IN HUMAN ORAL KERATINOCYTES BY FLOW CYTOMETRY AND DNA CELL CYCLE ANALYSIS... 

Mechanistic studies [35] showed combination treatments to inhibit cell proliferation via downregulation of cyclin D1 and induce apoptosis via activation of caspases-8 and -9 and downregulation of prosurvival proteins, FLIP and survivin [35]. Several stUhenes, related to resveratrol, have been synthesized and tested for their anticancer effect on HL-60 leukemia cell line, taking particular care of the cell cycle analysis. Figure 8.2 shows synthesis of stilbenes and its effects on cell cyde. A scheme of flow cytometry analysis of cell cycle is presented in Figure 8.3. The most potent compound was found to be (Z)-3,4, 5-trimethoxystilbene(I)... 

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. ... 

Ormerod, M. G. Kubbies, M. Cell cycle analysis of asynchronous cell populations by flow cytometry using bromodeoxyuridine label and Hoechst-propidium iodide stain. Cytometry 1992, 13, 678-h85. 

Rabinovitch, P. S. Kubbies, M. Chen, Y. C. Schindler, D. Hoehn, H. BrdU-Hoechst flow cytometry a unique tool for quantitative cell cycle analysis. Exp. Cell Res. 1988,174, 309-318. 

Flow cytometry (FCM) is widely used for exploring mechanism of action of compounds that compromise proliferation since it is rapid, accurate and usable for any cellular context [5], In this chapter we want to point out technical and strategic aspects of use of FCM for cell cycle studies of a putative anticancer agent. As an example we used Edotecarin, a topi inhibitor, firstly evaluating proliferation outcome and classical DNA content analysis by propidium iodide, and then since the compound treatment produced cell cycle perturbation difficult to interprete, a two-parametric analysis by 5-bromo-deoxyuridine (BrdU) was applied for separating cell cycle phases. Moreover we put our efforts into identifing specific cell cycle arrest not easily demonstrable by previously described methods, through the use of in vitro kinetics ( pulse and chase ). Finally, in vivo assessment of efficacy and biomarkers modulation after treatment was analyzed. 

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),... 

So, these results showed a double effect of BOA on lettuce meristems an increasing significant delay in the cell cycle progression and a decrease in the mitotic index. Flow cytometry analysis showed a weak effect of BOA at cell cycle level in the step from G2 to M. BOA effect appeared to retard the cell cycle progression of treated-root meristems, which was very clear after 10 h BOA exposition (Fig. 12.5), and as an inhibition of the number of cells undergoing cell division. 

The effects exhibited in the flow cytometry analysis are also clearly detected by the mitotic index technique (unpublished data), which appears to be an excellent complementary technique in the cell cycle studies using flow cytometry. The mitotic index (see Fig. 12.5) revealed that the cell cycle progression goes slower in BOA-treated meristems than in control meristems and also that the maximum number of cells undergoing cell division is fewer and later in treated meristems (18% at 6 h BOA exposure time) than in control meristems (36% at 4 h distilled water exposure time). 

Flow cytometry is a very versatile technique [223] which allows the analysis of more than 104 cells per second [369,370]. This high number results in statistically significant data and distributions of cell properties. Therefore, flow cytometry is a key technique to segregate biomass (into distinct cell classes) and to study microbial populations and their dynamics, specifically the cell cycle [76, 87, 116, 200, 214, 221, 295, 329, 330, 409, 418]. Individual cells are aligned by means of controlled hydrodynamic flow patterns and pass the measuring cell one by one. One or more light sources, typically laser(s), are focused onto the stream of cells and a detection unit(s) measure(s) the scattered and/or fluorescent light (Fig. 24). Properties of whole cells such as size and shape can be... 

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