Laser scanning cytometry (LCS) allows detailed analysis of the cell cycle in PI stained human fibroblasts (TIG‐7)

Kawasaki, Masahiro; Sasaki, Kohsuke; Satoh, Takatomo; Kurose, Akira; Kamada, Tomomi; Furuya, Tomoko; Murakami, Tomoyuki; Todoroki, Takeshi

doi:10.1111/j.1365-2184.1997.tb00930.x

Cited by 30 publications

(19 citation statements)

References 13 publications

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“…Laser scanning cytometry (LSC), a technique based on a microscope cytofluorimeter, 13 was also used for cell proliferation analysis purposes. [14][15][16] At present, few techniques allow a highcontent analysis of cell proliferation at the single cell level and simultaneous acquisition of morphological data.…”

Section: Introductionmentioning

confidence: 99%

Quantification of the Proliferation Index of Human Dermal Fibroblast Cultures with the ArrayScan™ High-Content Screening Reader

Gasparri¹,

Mariani²,

Sola³

et al. 2004

SLAS Discovery

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High-throughput cell-based assays are becoming a powerful approach in the drug discovery process. The ArrayScan high-content screening (HCS) reader is a cytometer based on a fully automated fluorescence microscope that is able to obtain quantitative information on the intensity and localization of fluorescence signals within single cells over a wide cell population. The aim of this work was to set up an automated HCS multiparameter analysis for the quantification of the in vitro proliferation index of normal human dermal fibroblast (NHDF) cultures. The authors stimulated starved NHDF with insulin-like growth factor-1, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, or serum, and they quantified the proliferation index by measuring the expression of Ki-67 antigen, the incorporation of bromodeoxyuridine (BrdU), and the phosphorylation of the retinoblastoma protein (pRb). This approach also allowed quantification of the mitotic index by phospho-histone H3 staining and the percentage of cells in the S-phase by BrdU incorporation. The proliferation data from the ArrayScan assays were validated by comparison with a reference enzyme-linked immunosorbent assay (ELISA) and by flow cytometry. The measured proliferation indices were highly reproducible in repeated measures and independent experiments. The authors therefore propose that the ArrayScan HCS system could be used for high-throughput multiparameter analysis and quantification of the proliferation of cellular cultures.

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Section: Introductionmentioning

confidence: 99%

Quantification of the Proliferation Index of Human Dermal Fibroblast Cultures with the ArrayScan™ High-Content Screening Reader

Gasparri¹,

Mariani²,

Sola³

et al. 2004

SLAS Discovery

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“…1). There were more stained particles (3)(4)(5)(6) scattered in the nuclei of corneal and conjunctival epithelial cells, especially in the superficial layers. The limbal superficial cells contained fewer AO-stained particles (1-3) than corneal and conjunctival superficial cells.…”

Section: Resultsmentioning

confidence: 98%

“…With appropriate excitation and emission filters, the fluorescence can be detected by CLSMs, and it has been shown that AO could be used for the identification of different kinds of cells and differentiation status, whereas Cal-AM and EthD-1 could be used to identify the viability of cells. [3][4][5][6][7][8][9] The ocular surface epithelium, which functions as a barrier, includes corneal, limbal, and conjunctival epithelium. It is of surface ectoderm origin and has generally similar structure in 1) the basal cell layer (with cells that gradually become thinner and larger more anteriorly) and 2) the wing cell layer and 3) the superficial cell layer (in which cells are terminally differentiated).…”

mentioning

confidence: 99%

Corneal, Limbal, and Conjunctival Epithelium of Bovine Eyes Imaged In Vitro by Using a Confocal Laser Scanning Microscope

Feng

Bantseev²,

Simpson³

2008

Cornea

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Confocal results of the corneal central, limbal, and conjunctival morphology are similar to those found in traditional microscopic observations. Bovine central corneal epithelium is thicker than limbal epithelium. However, the nuclear AO staining pattern of unfixed ocular surface epithelium of bovine eyes in vitro might represent the cell differentiation status. With the aid of the fluorescence dye, confocal laser scanning microscopy can provide unique morphometric information about corneal, limbal, and conjunctival epithelial cells.

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“…Note the decrease in PI fluorescence, which is more pronounced in PHA stimulated lymphocytes, following treatment with RNase. Analysis of PI fluorescence area versus maximal pixel of the RNase-treated cells (G,H) makes it possible to distinguish G 0 and M lymphocytes that have condensed chromatin (high maximal pixel, low area) from the interphase G 1 , S, and G 2 cells, as described before (17)(18)(19)(20). Cell gallery (right panels) shows the respective G 0 , G 1 , S, G 2 , and M cells relocated after the measurement of their fluorescence by LSC.…”

Section: Staining Of Cellular Dna Rna and Protein: Fluorescence Meamentioning

confidence: 99%

“…The bivariate distributions of PI fluorescence area versus maximal pixel reveal the degree of nuclear chromatin condensation. For cells with the same DNA content, the more condensed their chromatin, the higher the intensity of maximal pixel of the DNA-associated fluorescence and the smaller the fluorescence area (17)(18)(19)(20). As shown in Figures 1G,H, the G 0 lymphocytes from the nonstimulated cultures and the M cells from PHA-stimulated cultures had a distinctly higher maximal pixel and lower fluorescence area compared with their counterparts with the same DNA content but with less condensed chromatin, the G 1 and G 2 cells.…”

Section: Staining Of Cellular Dna Rna and Protein: Fluorescence Meamentioning

confidence: 99%

Bivariate analysis of cellular DNA versus RNA content by laser scanning cytometry using the product of signal subtraction (differential fluorescence) as a separate parameter

Smolewski

Grabarek

Kamentsky³

et al. 2001

Cytometry

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Background:The cytometric methods of bivariate analysis of cellular RNA versus DNA content have limitations. The method based on the use of metachromatic fluorochrome acridine orange (AO) requires rigorous conditions of the equilibrium staining whereas pyronin Y and Hoechst 33342 necessitate the use of an instrument that provides two-laser excitation, including the ultraviolet (UV) light wavelength. Methods: Phytohemagglutinin (PHA)-stimulated human lymphocytes were deposited on microscope slides and fixed. DNA and double-stranded (ds) RNA were stained with propidium iodide (PI) and protein was stained with BODIPY 630/650-X or fluorescein isothiocyanate (FITC). Cellular fluorescence was measured with a laser scanning cytometer (LSC). The cells were treated with RNase A and their fluorescence was measured again. The file-merge feature of the LSC was used to record the cell PI fluorescence measurements prior to and after the RNase treatment in list mode, as a single file. The integrated PI fluorescence intensity of each cell after RNase treatment was subtracted from the fluorescence intensity of the same cell measured prior to RNase treatment. This RNase-specific differential value of fluorescence (differential fluorescence [DF]) was plotted against the cell fluorescence measured after RNase treatment or against the protein-associated BODIPY 630/650-X or FITC fluorescence. Results: The scattergrams were characteristic of the RNA versus DNA bivariate distributions where DF represented cellular ds RNA content and fluorescence intensity of the RNase-treated cells, their DNA content. The distributions were used to correlate cellular ds RNA content with the cell cycle position or with protein content. Conclusions: One advantage of this novel approach based on the recording and plotting of DF is that only the RNase -specific fraction of cell fluorescence is measured with no contribution of nonspecific components (e.g., due to the emission spectrum overlap or stainability of other than RNA cell constituents). Another advantage is the method's simplicity, which ensues from the use of a single dye, the same illumination, and the same emission wavelength detection sensor for measurement of both DNA and ds RNA. The method can be extended for multiparameter analysis of cell populations stained with other fluorochromes of the same-wavelength emission but targeted (e.g., immunocytochemically) for different cell constituents. Cytometry 45: 73-78, 2001.

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Laser scanning cytometry (LCS) allows detailed analysis of the cell cycle in PI stained human fibroblasts (TIG‐7)

Cited by 30 publications

References 13 publications

Quantification of the Proliferation Index of Human Dermal Fibroblast Cultures with the ArrayScan™ High-Content Screening Reader

Quantification of the Proliferation Index of Human Dermal Fibroblast Cultures with the ArrayScan™ High-Content Screening Reader

Corneal, Limbal, and Conjunctival Epithelium of Bovine Eyes Imaged In Vitro by Using a Confocal Laser Scanning Microscope

Bivariate analysis of cellular DNA versus RNA content by laser scanning cytometry using the product of signal subtraction (differential fluorescence) as a separate parameter

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