Methods for Determining the Proliferation Kinetics of Cells by Means of 5‐bromodeoxyuridine

Pera, Franz; Mattias, P.; Detzer, K.

doi:10.1111/j.1365-2184.1977.tb00293.x

Cited by 17 publications

(14 citation statements)

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“…The proliferation fraction can be estimated either by measuring the DNA content per cell (with cells in division having more DNA than resting cells) or by a two parameter, RNA-DNA content analysis. In essence, commitment to division is associated with upregulation of RNA expression followed by DNA synthesis (Pera et al, 1977;Bohmer, 1979;Kubbies et al, 1992;Stout and Suttles, 1992;Veiga-Fernandes et al, 2000). As we show in the Appendix (Section A.2.3), asymptotically the proliferation fraction P is given by…”

Section: Indirect Fittingmentioning

confidence: 96%

“…Measuring an additional parameter, the fraction of cells in division P (i.e., fraction of cells that are in the S + G 2 + M phase of the cell cycle) may allow estimation of all parameters of the SM model (Table 6). The fraction of cells in the S + G 2 + M phase of the cell cycle can be estimated by looking at either DNA content per cell (for example, using PI staining) or by a two parameter, RNA-DNA content analysis (Pera et al, 1977;Bohmer, 1979;Kubbies et al, 1992;Stout and Suttles, 1992;VeigaFernandes et al, 2000).…”

Section: Quantifying Cell Turnovermentioning

confidence: 99%

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Quantifying cell turnover using CFSE data

Ganusov

Pilyugin

Boer

et al. 2005

Journal of Immunological Methods

120

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The CFSE dye dilution assay is widely used to determine the number of divisions a given CFSE labelled cell has undergone in vitro and in vivo. In this paper, we consider how the data obtained with the use of CFSE (CFSE data) can be used to estimate the parameters determining cell division and death. For a homogeneous cell population (i.e., a population with the parameters for cell division and death being independent of time and the number of divisions cells have undergone), we consider a specific biologically based bSmith-MartinQ model of cell turnover and analyze three different techniques for estimation of its parameters: direct fitting, indirect fitting and rescaling method. We find that using only CFSE data, the duration of the division phase (i.e., approximately the S + G 2 + M phase of the cell cycle) can be estimated with the use of either technique. In some cases, the average division or cell cycle time can be estimated using the direct fitting of the model solution to the data or by using the Gett-Hodgkin method [Gett A. and Hodgkin, P. 2000. A cellular calculus for signal integration by T cells. Nat. Immunol. 1:239-244]. Estimation of the death rates during commitment to division (i.e., approximately the G 1 phase of the cell cycle) and during the division phase may not be feasible with the use of only CFSE data. We propose that measuring an additional parameter, the fraction of cells in division, may allow estimation of all model parameters including the death rates during different stages of the cell cycle. D

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Section: Indirect Fittingmentioning

confidence: 96%

Section: Quantifying Cell Turnovermentioning

confidence: 99%

Quantifying cell turnover using CFSE data

Ganusov

Pilyugin

Boer

et al. 2005

Journal of Immunological Methods

120

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“…We think that blocking the endogenous synthesis of dThd with FdUrd will not only improve the quantitative BrdUrd technique in cell kinetic studies by flow cytometry, but may also be useful for the measurement of proliferation kinetics with BrdUrd-labeled cells after Hfluorescence staining (9,22) plus Giemsa staining (30). In this context, it is noteworthy that the application of FdUrd for the inhibition of endogenous dThd synthesis is a routine procedure in caesium chloride buoyant density studies using BrdUrd (34).…”

Section: Discussionmentioning

confidence: 99%

Effect of 5‐fluoro‐2′‐deoxyuridine (FdUrd) on 5‐bromo‐2′‐deoxyuridine (BrdUrd) incorporation into DNA, measured with a monoclonal BrdUrd antibody and by the BrdUrd/hoechst quenching effect

Ellwart

Dormer

1985

Cytometry

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The purpose of this study was to improve the application of bromodeoxyuridine (BrdUrd) for the flow cytometric analysis of cell kinetics. In order to obtain a quantitative measure of the DNA synthesis rate (or the number of divided cells), BrdUrd should replace thymidine (dThd) completely in the newly synthesized DNA strands. The de novo synthesis of dThd monophosphate competing with BrdUrd incorporation was stopped by fluorodeoxyuridine (FdUrd). Cells of a human leukemic cell line (REH) were exposed 4.0 BrdUrd for either 20 min, 8 h, or 24 h. Bromodeoxyuridine incorporation was determined by a monoclonal antibody as well as by the BrdUrd/Hoechst (HI technique. Counterstaining of the DNA was performed with propidium iodide or ethidium bromide. DNA fluorescences were measured in both techniques with a two-parameter flow cytometer, the histograms being analyzed by computer. It was found that FdUrd is required in the BrdUrd/ H technique for replacement of dThd at low BrdUrd concentrations and long incubation times. With short incubation periods, as used for detection by the monoclonal anti-BrdUrd antibody, FdUrd increases the incorporated BrdUrd amount when BrdUrd concentrations of 10 pM or less are applied.Key terms: Bromodeoxyuridine, fluorodeoxyuridine, DNA synthesis rate, human leukemic cell line, flow cytometry Bromodeoxyuridine (BrdUrd) is used with increasing frequency in cell kinetic studies after the introduction of the Hoechst (H) quenching effect into flow cytometry by Latt (23,241. Bohmer (2) and Kubbies and Rabinovitch (20) described methods of cell cycle analysis using the quenching of H in divided cells. Perturbations of the cell cycle of normal and tumor cells were easy to investigate with the BrdUrdM technique (1,29). Counterstaining of total DNA per cell with ethidium bromide (EB) simplified the analysis of the DNA synthesis in the first cycle (3,4,16). This two-parameter technique allows flow cytometric measurement of the DNA synthesis rate even in subcompartments of the S-phase after a few hours of BrdUrd incubation (28,151). Since it is now possible to measure DNA synthesis with the monoclonal anti-BrdUrd antibody (10,18,26), BrdUrd is being applied increasingly for cell cycle studies. It is therefore of interest to know the exact conditions of quantitative BrdUrd incorporation.The quantity of BrdUrd instead of thymidine (dThd) incorporated per unit of time into DNA may provide information on the DNA synthesis rate. For quantitative assessment of the DNA synthesis rate, it is most convenient if BrdUrd replaces dThd completely in the newly synthesized DNA strands. It is, however, to be expected that the endogenous synthesis of the DNA precursor dThd monophosphate competes with the exogenously supplied BrdUrd. In addition, an intracellular precursor pool of dThd monophosphate exists at the beginning of BrdUrd incorporation (7) and should not mix with the newly synthesized BrdUrd monophosphate. Dormer applied fluorodeoxyuridine (FdUrd) to block the endogenous synthesis of dThd monophosphate for us...

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“…Thus, analysis of the cellular kinetics will produce important information enabling researchers to better understand the causes of disease about what is occurring in the tissues. The immunohistochemical technique using an antibody to recognize BrdU, a thymidine analogue, is a conventional method used to detect cell proliferation [5,24,32]. BrdU is incorporated into the cells only during the S phase, i.e.…”

Section: Discussionmentioning

confidence: 99%

Localization of Proliferative and Apoptotic Cells in the Kidneys of ICR-Derived Glomerulonephritis(ICGN) Mice.

Yamaguchi

Manabe

Uchio‐Yamada

et al. 2001

The Journal of Veterinary Medical Science

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ABSTRACT. The ICR-derived glomerulonephritis (ICGN) mouse is a novel inbred mouse strain with a hereditary nephrotic syndrome, considered to be a good model of human idiopathic nephrotic syndrome and develops proteinuria, hypoproteinemia and anemia. In the present study, we compared the cell kinetics in the kidneys of ICGN mice with age-matched ICR mice as normal controls. The proliferating cells were visualized by 5-bromo-2'-deoxyuridine labeling, and apoptotic cells were determined by terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling. Many proliferating epithelial cells of renal tubules, glomerular mesangial cells and tublointerstitial fibroblast-like cells were observed in the kidneys of ICGN mice, but no proliferating cells were seen in the kidneys of ICR mice. Apoptotic cells had round nuclei, and were observed only in the tubulointerstitium in the kidneys of ICGN mice but not in that of controls. The proliferation of renal tubular epithelial cells may represent a compensatory response, and that of mesangial and fibroblast-like cells may play a pathogenic role in nephrotic syndrome. Apoptosis in tubulointerstitial cells with round nuclei may have been erythropoietin-producing cells, and probably caused anemia. KEY WORDS: apoptotic cell, hereditary nephrotic mouse (ICGN), kidney, proliferating cell.J. Vet. Med. Sci. 63 (7): [781][782][783][784][785][786][787] 2001 The ICR-derived glomerulonephritis (ICGN) mouse is a novel inbred mouse strain with a hereditary nephrotic syndrome of unknown etiology, and considered to be a good model of human idiopathic nephrotic syndrome. ICGN mice show proteinuria at a young age, later develop hypoproteinemia, hyperlipemia and anemia, and approximately 40% of them fall into severe systemic edema [17][18][19][20][21][22]. Our previous studies [28,29] showed that many kinds of extracellular matrix (ECM) components, both basement membrane and interstitial components, abnormally accumulated in glomeruli and tubulointerstitium of ICGN mouse kidneys. We also reported that the progression of fibrotic degeneration in ICGN mouse kidneys may be caused by overproduction of ECM components, inhibition of ECM breakdown, and decreased activities of matrix metalloproteinases [30]. However, the cell kinetics about which types of cells proliferate and/or die in ICGN mouse kidneys have not been investigated. To understand the pathogenic mechanism of nephrotic and fibrogenic degeneration in the kidneys of ICGN mice, it is essential to clarify the timedependent changes in the cell kinetics of their kidneys. In the present study, we compared the cell kinetics in the kidneys of ICGN mice with age-matched ICR mice as normal controls. The proliferating cells were visualized by 5-bromo-2'-deoxyuridine (BrdU) labeling, and apoptotic cells were histochemically determined by terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL). MATERIALS AND METHODS Animals and tissue preparation:Nep...

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Methods for Determining the Proliferation Kinetics of Cells by Means of 5‐bromodeoxyuridine

Cited by 17 publications

References 9 publications

Quantifying cell turnover using CFSE data

Quantifying cell turnover using CFSE data

Effect of 5‐fluoro‐2′‐deoxyuridine (FdUrd) on 5‐bromo‐2′‐deoxyuridine (BrdUrd) incorporation into DNA, measured with a monoclonal BrdUrd antibody and by the BrdUrd/hoechst quenching effect

Localization of Proliferative and Apoptotic Cells in the Kidneys of ICR-Derived Glomerulonephritis(ICGN) Mice.

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