Flow cell-cycle analysis

For cell cycle analysis, HOS cells (1 x 106) were treated with the indicated concentrations (see Table 13.2) of MTX or MTX-LDH for 20h, harvested by trypsin treatment, washed with PBS, and then fixed with cold 70 % ethanol on ice overnight. The fixed cells were incubated with propidium iodide and flow cytometric measurement was carried out. Over 20 h, an increase in the number of cells in the Gl phase resulted from MTX and MTX-LDH treatment compared to untreated cells, indicating arrest at the Gl/S boundary. It is worth noting that the inhibition of the Gl/S transition was more evident in the cells treated with MTX-LDH than in those treated with free MTX (85.59% versus 66.62% at 320pM/ml). This is consistent with... 

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... 

Additionally, two studies have measured colorectal epithelial cell proliferation and apoptosis in human non-neoplastic mucosa in combination with serum bile acid quantification. Ochsenkuhn et al have reported a positive correlation between serum DCA levels and proliferation measured by flow cytometric cell cycle analysis. However, a more recent study of colorectal adenoma patients failed to detect a correlation between serum DCA and immuno-histochemical Ki-67 antigen labelling. Instead, this latter study revealed a positive correlation between serum DCA and the degree of TUNEL-positive epithelial cell apoptosis. ... 

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. 9. The cell cycle. M mitosis G1+S+G2 interphase, i.e., the period between ceU division. During G1 (G for gap) the cellular genome is in the diploid state. This is followed by the S-phase (S for synthesis) during which the DNA is replicated, and finally G2. Insert Cell-cycle analysis of HM02 cells cultured with and without vinblastine. CeU-cycle distribution was determined by staining DNA with propidium iodide, and the number of cells in different phases of the cell cycle was measured with a FACStar flow cytometer. Vinblastine, which disrupts the formation of microtubules, causes cell-cycle arrest in the G2/M-phase...

Dolbeare, F, Kuo, W. L., Vanderlaan, M., and Gray, J W (1988) Cell cycle analysis by flow cytometric analysis of the incorporation of lododeoxyuridme (IdUrd) and bromodeoxyuridine (BrdUrd) Proc Am Assoc Cancer Res 29, 1896-1901... 

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),... 

At the cellular level cisplatin causes a block in the cell cycle at the point where DNA repair occurs, the S/G2 interface. The stages of the cell cycle can be monitored using flow cytometry75. In this technique cells are fixed and stained with propidium iodide, a DNA fluorochrome, which binds quantitatively to DNA. The degree of fluorescence is therefore directly proportional to the DNA content of the cell, which in turn is an indicator of the position of the cells in the cell cycle. The ovarian cell line, SK-OV-3, was incubated with [Au(acetato)2(damp)] for 4 hours at concentrations of either 30 pM or 100 pM and cell cycle analysis determined from DNA content. Using this technique it was apparent that the gold(III) complex [Au(OAc)2(damp)], unlike cisplatin, did not exert a cell cycle specific effect69. 

DiFrancesco, L.M., Murthy, S.K., Luider, J., and Demetrick, D.J. (2000) Laser capture microdissection-guided fluorescence in situ hybridization and flow cytometric cell cycle analysis of purified nuclei from paraffin sections. Mod Pathol 13, 705-11. 

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. 

Poot, M. Hoehn, H. Kubbies, M. Grossmatm, A. Chen, Y. Rabinovitch, P. S. Cell cycle analysis using continuous bromodeoxyuridine labeling and Hoechst 33258-ethidium bromide bivariate flow C5 ometry. Methods Cell Biol. 1990,55,185-198. 

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. 

EC-ALE studies of ZnTe using a TLEC were performed with up to 20 cycles of deposition [130], Coulometry was the only analysis performed on the deposits. A plot of coverage as a function of the number of cycles was linear, as expected for a surface limited process. No thicker films have as yet been formed using the flow deposition system. At present there is no reason to believe that the cycle developed for 20 cycle deposits will not produce good quality deposits of any given thickness, using the automated flow-cell system. 

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. 

Unfortunately monoparametric DNA content analysis by PI is not able to discriminate from different cell cycle phases, and as exemplified in Figure 4, monoparametric analysis by PI show its limitations, since this flow cytometric assay does not display any details S-phase activities after drug treatments. At this point, cells can be arrested in a specific cell phase (eg. Gi or G2/M) and obviously a decrease of S-phase is observed. 

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