Cell cycle analysis in vitro using flow cytofluorimetry after synchronization

Watson, James V.; Taylor, Ian W.

doi:10.1038/bjc.1977.188

Cited by 12 publications

(12 citation statements)

References 16 publications

(15 reference statements)

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“…It makes no assumptions concerning the age distribution of the population (22), which was one of the problems associated with our methods published previously (25,28), and no assumptions about the rate of DNA synthesis or of the G2 +M:G1 ratio. The latter should obviously be 2.0; however, we only estimate DNA indirectly by flow cytometry.…”

Section: Discussionmentioning

confidence: 99%

A pragmatic approach to the analysis of DNA histograms with a definable G1 peak

1987

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A method for DNA histogram analysis is described that depends only on the simple assumption that the data are normally distributed and a requirement that a G1 peak is present. A probability density function was derived from the assumption that extracted the S-phase component from the whole histogram. The model was tested with simulated data, and good agreement between predicted and known proportions in G1, S, and G1+M was found. Good agreement was also found between duplicates of experimentally derived data. Some systematic errors are present in the analysis of certain types of histograms. However, these result in small errors when compared with biological and experimental variation and are less than the average of algorithms in current use.The program required only two queued requests, those of the start and the end channels over which the analysis is to be performed. The algorithms perform rapidly on a microcomputer with only 28K addressable memory. Only two failures occurred in over 350 analyses and the method can be used for drug-and radiation-perturbed populations as well as with unperturbed. Key terms: Flow cytometry, DNA histogram analysisA number of models have been proposed for the analysis of flow cytometric DNA histogram data (1, 2, 4-9, 11-18, 25, 28-29) and a comparative review of the various methods has been published (3). .As expected, some models performed better than others, not only with simulated but also with experimentally derived data; however, none was ideal. The models that performed best tended to be those requiring a large mainframe computer, as they generally contained a large number of variables for which a solution had to be found. These include the position of the mean of the G1 and G2+M peaks, the standard deviations of these peaks, the age distribution of the population (22), the rate of DNA synthesis, and the relative durations of the G1, S, and G2 +M phase times with their standard deviations. Thus far, 12 variables have been defined, all of which may have to be considered simultaneously, which is not a trivial task, even with a large computer. Herein lies one of the central problems. The experimental DNA histogram contains, at best, two peaks and a trough corresponding to G1, G2 +M, and S-phase, respectively. This is a very simple data set with which to compute 12 variables, and this must be totally inadequate compared with the complexity of the biology.The work presented in this paper was undertaken to simplify the analysis of DNA histograms and to produce a robust method that gives results rapidly (within 30 s) on a microcomputer with only 28K addressable memory. THEORY AssumptionsIn our attempt to produce a "minimum assumption and computing" model (MAC) we have only assumed that the data are normally distributed. The Computer ProgramThe method of analysis will be illustrated by a description of the program flow diagram and the components of its various steps.Step 1. Eliminate high-frequency noise, if this exists, by "spreading" the data with a constant standard deviation o...

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Section: Discussionmentioning

confidence: 99%

A pragmatic approach to the analysis of DNA histograms with a definable G1 peak

1987

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“…The method of flow microfluorornetry allows a physician to obtain information on cell cycle patterns in an individual tumor. For further discussion of the technique and its applications we refer the reader to Dean and Jett (1974), Fried (1977), Haanen, Hillen, andWessels (1979, Watson andTaylor (1977), and Watson (1977).…”

Section: Dna Measurementsmentioning

confidence: 99%

Deconvolution of Microfluorometric Histograms withBSplines

Mendelsohn¹,

Rice

1982

Journal of the American Statistical Association

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We consider the problem of estimating a probability density from observations from that density which are further contaminated by random errors. We propose a method of estimation using spline functions, discuss the numerical implementation of the method, and prove its consistency. The problem is motivated by the analysis of DNA content obtained by microfluorometry , and an example of such an analysis is included.

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“…Mitotic selection (Teresima & Tolmach, 1963) was employed as the synchronizing event. Details of our methods have been described previously (Watson & Taylor, 1977) but, briefly, cells in mitosis were shaken from the monolayers of six 150 cmz flasks containing exponentially growing cells. The selected cells were pooled and reseeded into three 25 cm2 plastic flasks.…”

Section: Emt6/m/cc Cellsmentioning

confidence: 99%

Nucleic Acid Profile of the Emt6 Cell Cycle In Vitro

Watson

Chambers

1978

Cell Proliferation

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Simultaneous RNA and DNA estimations were carried out during the cell cycle of EMT6/M/CC cells growing in vitro following synchronization by mitotic selection. the determinations were performed with a flow cytofluorimeter on individual cells stained with acridine orange. It was found that the RNA content increased during G1 then remained virtually constant between early and mid S phase, but a second increase occurred during late S. the rate of uptake of tritiated uridine paralleled these changes in RNA levels, and it was also found that the rate of uptake in metaphase and anaphase was virtually zero, but a rapid increase occurred in telophase. the increase in DNA during S was approximately linear, and the intermitotic phase and cycle durations were very similar to previously reported results.

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Cell cycle analysis in vitro using flow cytofluorimetry after synchronization

Cited by 12 publications

References 16 publications

A pragmatic approach to the analysis of DNA histograms with a definable G1 peak

A pragmatic approach to the analysis of DNA histograms with a definable G1 peak

Deconvolution of Microfluorometric Histograms withBSplines

Nucleic Acid Profile of the Emt6 Cell Cycle In Vitro

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