Experimental reconsideration of the utility of serum starvation as a method for synchronizing mammalian cells

Cooper, Stephen; Gonzalez-Hernandez, Mariam B.

doi:10.1016/j.cellbi.2008.09.009

Cited by 17 publications

(21 citation statements)

References 33 publications

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“…Many experimental approaches have been described for synchronizing cells at specific phases of the cell cycle [15], and several common methods involve pharmacological agents acting at various points throughout the cell cycle [16], [17]. However, because of adverse cellular perturbations that can result from exposure to these pharmacological agents [18], we chose to use serum deprivation as the method for synchronizing undifferentiated THP-1 cells.…”

Section: Resultsmentioning

confidence: 99%

Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

2013

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The human THP-1 cell line is widely used as an in vitro model system for studying macrophage differentiation and function. Conventional culture conditions for these cells consist of ambient oxygen pressure (∼20% v/v) and medium supplemented with the thiol 2-mercaptoethanol (2-ME) and serum. In consideration of the redox activities of O2 and 2-ME, and the extensive experimental evidence supporting a role for reactive oxygen species (ROS) in the differentiation and function of macrophages, we addressed the question of whether culturing THP-1 cells under a more physiologically relevant oxygen tension (5% O2) in the absence of 2-ME and serum would alter THP-1 cell physiology. Comparisons of cultures maintained in 18% O2 versus 5% O2 indicated that reducing oxygen tension had no effect on the proliferation of undifferentiated THP-1 cells. However, decreasing the oxygen tension to 5% O2 significantly increased the rate of phorbol ester-induced differentiation of THP-1 cells into macrophage-like cells as well as the metabolic activity of both undifferentiated and PMA-differentiated THP-1 cells. Removal of both 2-ME and serum from the medium decreased the proliferation of undifferentiated THP-1 cells but increased metabolic activity and the rate of differentiation under either oxygen tension. In differentiated THP-1 cells, lowering the oxygen tension to 5% O2 decreased phagocytic activity, the constitutive release of β-hexosaminidase and LPS-induced NF-κB activation but enhanced LPS-stimulated release of cytokines. Collectively, these data demonstrate that oxygen tension influences THP-1 cell differentiation and primary macrophage functions, and suggest that culturing these cells under tightly regulated oxygen tension in the absence of exogenous reducing agent and serum is likely to provide a physiologically relevant baseline from which to study the role of the local redox environment in regulating THP-1 cell physiology.

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Section: Resultsmentioning

confidence: 99%

Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

2013

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“…Our model system of FL5.12 cells in response to IL-3 is perhaps a superior model for the G0/G1 transition to the more common serum-starvation and refeeding experiments that carry with them the complexity of signals in serum and the concerns of synchronization and re-activation (Cooper and Gonzalez-Hernandez, 2009). While there still exists a significant fraction of cells that loses synchrony in our system along the cell cycle, a significant sub-population of cells remarkably gave rise to major known characteristics and features of each cell cycle phase as evidenced in Figs.…”

Section: Discussionmentioning

confidence: 99%

Proteomic and Metabolomic Characterization of a Mammalian Cellular Transition from Quiescence to Proliferation

et al. 2017

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SUMMARY There exist similarities and differences in metabolism and physiology between normal proliferative cells and tumor cells. Once a cell enters the cell cycle, metabolic machinery is engaged to facilitate various processes. The kinetics and regulation of these metabolic changes have not been properly evaluated. To correlate the orchestration of these processes with the cell cycle, we analyzed the transition from quiescence to proliferation of a non-malignant murine pro-B lymphocyte cell line in response to IL-3. Using multiplex mass-spectrometry-based proteomics we show that the transition to proliferation shares features generally attributed to cancer cells: up-regulation of glycolysis, lipid metabolism, amino-acid synthesis, and nucleotide synthesis and down-regulation of oxidative phosphorylation and the urea cycle. Furthermore, metabolomic profiling of this transition reveals similarities to cancer-related metabolic pathways. In particular, we find that methionine is consumed at a higher rate than other essential amino acids, with a potential link to epigenetic maintenance.

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“…To the many critiques of the Standard Model-that methods used to synchronize cells do not synchronize cells [13,[19][20][21][22][23][24][25][26][27][28][29], that data in support of the standard model have been shown to be not reproducible [15,16], that gene expression variations cannot have any significant affect on passage through the cell cycle [17], that logical problems with the Standard Model have not been addressed [11]-I now add a critique of the use of normalization to present data on gene expression during the division cycle. …”

Section: The Continuum Modelmentioning

confidence: 99%

“…Whole-culture inhibition methods where cells are growth inhibited and where it is proposed that the cells are synchronized upon release of inhibition do not synchronize cells. These methods have been shown to not synchronize cells by both theoretical analysis [19,23,24] and by numerous experimental analyses [21,[27][28][29]. Even publications that propose that methods such as serum starvation or thymidine inhibition can synchronize cells, these papers often present data that these methods do not synchronize cells [1] when considered in the light of clearly defined criteria for synchronization [19].…”

mentioning

confidence: 99%

“…The data supporting the current, consensus, dominant, widely-cited, and well-recognized Standard Model stem mostly from the most used and flawed approach to cell-cycle analysis, the use of whole-culture synchronization methods that do not synchronize cells [13,[19][20][21][22][23][24][25][26][27][28][29]. The popular methods used for "synchronizing" cells are not criticized here because they synchronize cells poorly, or only occasionally, or that synchrony does not last, but because these batch or whole-culture inhibition methods do not synchronize cells at all.…”

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confidence: 99%

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Gene Expression during the Cell Cycle: Obfuscation of Original Cell-Cycle Gene Expression Data by Normalization

Cooper

2015

Journal of Cells

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Normalization of raw data on gene expression during the cell cycle obscures the original experimental data and makes it appear as if all genes have the same numerical level of cyclical expression. Results that would not support gene expression because of minimal variations thus appear, after normalization, to be stronger than they actually are. Consideration of the effect of normalization raises critical questions about many experiments on the cell cycle dependent variation of gene expression-that is, proposed cyclical changes in mRNA content-during the cell cycle.Keywords: Cell-cycle, Normalization, Gene expression, mRNA synthesis. Contribution/ OriginalityThis paper demonstrates that normalization makes minimal variations appear much stronger than they are in the raw data, and thus one should be skeptical of some published results proposing cell-cycle specific gene expression.

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Experimental reconsideration of the utility of serum starvation as a method for synchronizing mammalian cells

Cited by 17 publications

References 33 publications

Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

Proteomic and Metabolomic Characterization of a Mammalian Cellular Transition from Quiescence to Proliferation

Gene Expression during the Cell Cycle: Obfuscation of Original Cell-Cycle Gene Expression Data by Normalization

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