Dataset Information

A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample.

ABSTRACT:

Background

Presently available flow cytometric methods of bromodeoxyuridine (BrdUrd) labelling do not provide information on the cell cycle time (TC) and the growth fraction (GF). In this paper, we describe a novel and simple method to estimate TC and GF from flow cytometric analysis of a single tumour sample after BrdUrd labelling.

Methods

The proposed method is based on two assumptions: (1) the number of labelled cells traversing the cell cycle per unit time is constant and (2) the total number of labelled cells is constant throughout the cycle, provided that cells produced after division are excluded. The total numbers of labelled divided G1 cells, labelled divided S cells, labelled undivided S cells, and labelled undivided G2 cells were obtained for DNA histograms of BrdUrd-positive cells in a collected sample. These cell numbers were used to write equations to determine the durations of cell cycle phases, TC and GF. To illustrate the application of the proposed formulae, cell cycle kinetic parameters were analysed in solid SL2 tumours growing in DBA/2 mice and in human T-leukaemia Jurkat cells in culture.

Results

The suitability of the proposed method for estimating durations of the cell cycle phases, TC and GF was demonstrated. TC in SL2 tumours was found to be relatively constant at 4 and 10 days after tumour implantation (20.3 +/- 1.1 h and 21.6 +/- 0.9 h, respectively). GF in tumours at day 10 was lower than GF at day 4 (54.2 +/- 7.7% vs. 79.2 +/- 5.9%, p = 0.0003). Approximate values of TC and GF of cultured Jurkat cells were 23.9 h and 79.3%, respectively.

Conclusion

The proposed method is relatively simple and permits estimation of the cell cycle parameters, including TC and GF, from a single tumour sample after labelling with BrdUrd. We have shown that this method may be useful in preclinical studies, allowing estimation of changes in GF during growth of murine tumours. Experiments with human Jurkat cells suggest that the proposed method might also prove suitable for measurement of cell kinetics in human tumours. Development of suitable software enabling more objective interpretation of the DNA profile in this method would be desirable.

SUBMITTER: Eidukevicius R

PROVIDER: S-EPMC1261259 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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Publications

A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample.

Eidukevicius Rimantas R Characiejus Dainius D Janavicius Ramunas R Kazlauskaite Nijole N Pasukoniene Vita V Mauricas Mykolas M Den Otter Willem W

BMC cancer 20050922

<h4>Background</h4>Presently available flow cytometric methods of bromodeoxyuridine (BrdUrd) labelling do not provide information on the cell cycle time (TC) and the growth fraction (GF). In this paper, we describe a novel and simple method to estimate TC and GF from flow cytometric analysis of a single tumour sample after BrdUrd labelling.<h4>Methods</h4>The proposed method is based on two assumptions: (1) the number of labelled cells traversing the cell cycle per unit time is constant and (2) ...[more]

PMID: 16176590

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Project description:Background and aimsFlow cytometry has been used to measure nuclear DNA content in pollen, mostly to understand pollen development and detect unreduced gametes. Published data have not always met the high-quality standards required for some applications, in part due to difficulties inherent in the extraction of nuclei. Here we describe a simple and relatively novel method for extracting pollen nuclei, involving the bursting of pollen through a nylon mesh, compare it with other methods and demonstrate its broad applicability and utility.MethodsThe method was tested across 80 species, 64 genera and 33 families, and the data were evaluated using established criteria for estimating genome size and analysing cell cycle. Filter bursting was directly compared with chopping in five species, yields were compared with published values for sonicated samples, and the method was applied by comparing genome size estimates for leaf and pollen nuclei in six species.Key resultsData quality met generally applied standards for estimating genome size in 81 % of species and the higher best practice standards for cell cycle analysis in 51 %. In 41 % of species we met the most stringent criterion of screening 10 000 pollen grains per sample. In direct comparison with two chopping techniques, our method produced better quality histograms with consistently higher nuclei yields, and yields were higher than previously published results for sonication. In three binucleate and three trinucleate species we found that pollen-based genome size estimates differed from leaf tissue estimates by 1·5 % or less when 1C pollen nuclei were used, while estimates from 2C generative nuclei differed from leaf estimates by up to 2·5 %.ConclusionsThe high success rate, ease of use and wide applicability of the filter bursting method show that this method can facilitate the use of pollen for estimating genome size and dramatically improve unreduced pollen production estimation with flow cytometry.

Dataset Information

A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample.

Background

Methods

Results

Conclusion

Publications

A method to estimate cell cycle time and growth fraction using bromodeoxyuridine-flow cytometry data from a single sample.

Similar Datasets

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