Density Separation of Quiescent Yeast Using Iodixanol

Quasem, Ishtiaque; Luby, Christopher J.; Mace, Charles R.; Fuchs, Stephen M.

doi:10.2144/000114596

Cited by 9 publications

(10 citation statements)

References 20 publications

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“…One limitation of this method is that it requires a large number of cells (2 × 10 9 ). Recently, a new density‐based separation approach was developed using iodixanol, for which the input cell number can be as low as 3 × 10 6 (Quasem, Luby, Mace, & Fuchs, 2017). An important caveat to these methods is that they enrich the quiescent population that is largely derived from new daughter cells as these cells are the smallest and most dense.…”

Section: Studying Quiescence In the Labmentioning

confidence: 99%

Cellular quiescence in budding yeast

Sun

Gresham

2021

Yeast

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Cellular quiescence, the temporary and reversible exit from proliferative growth, is the predominant state of all cells. However, our understanding of the biological processes and molecular mechanisms that underlie cell quiescence remains incomplete. As with the mitotic cell cycle, budding and fission yeast are preeminent model systems for studying cellular quiescence owing to their rich experimental toolboxes and the evolutionary conservation across eukaryotes of pathways and processes that control quiescence. Here, we review current knowledge of cell quiescence in budding yeast and how it pertains to cellular quiescence in other organisms, including multicellular animals. Quiescence entails large‐scale remodeling of virtually every cellular process, organelle, gene expression, and metabolic state that is executed dynamically as cells undergo the initiation, maintenance, and exit from quiescence. We review these major transitions, our current understanding of their molecular bases, and highlight unresolved questions. We summarize the primary methods employed for quiescence studies in yeast and discuss their relative merits. Understanding cell quiescence has important consequences for human disease as quiescent single‐celled microbes are notoriously difficult to kill and quiescent human cells play important roles in diseases such as cancer. We argue that research on cellular quiescence will be accelerated through the adoption of common criteria, and methods, for defining cell quiescence. An integrated approach to studying cell quiescence, and a focus on the behavior of individual cells, will yield new insights into the pathways and processes that underlie cell quiescence leading to a more complete understanding of the life cycle of cells. Take Away Quiescent cells are viable cells that have reversibly exited the cell cycle Quiescence is induced in response to a variety of nutrient starvation signals Quiescence is executed dynamically through three phases: initiation, maintenance, and exit Quiescence entails large‐scale remodeling of gene expression, organelles, and metabolism Single‐cell approaches are required to address heterogeneity among quiescent cells

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Section: Studying Quiescence In the Labmentioning

confidence: 99%

Cellular quiescence in budding yeast

Sun

Gresham

2021

Yeast

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“…We used a relatively low concentration (20%) of OptiPrep™ (a non‐ionic solution of 60% iodixanol) here, which has proved to be biocompatible, has low osmotic pressure and low intrinsic viscosity, and suitable for the culture of multiple types of cells in microdroplets (Brower et al, 2020; Hsu et al, 2018). Researchers have used 50% OptiPrep™ to isolate quiescent yeast cells, and the results show that the isolated cells have growth behaviour similar to untreated cells and remain resist to heat shock and zymolyase treatment (Quasem et al, 2017). Here, we used the addition of 20% OptiPrep™ to reduce the effect of cell sedimentation (the density of yeast cells is 1.1 g/ml, which is higher than that of culture medium), and to temporarily create neutrally buoyant cell suspensions without noticeable adverse effects.…”

Section: Resultsmentioning

confidence: 99%

“…We used a relatively low concentration (20%) of OptiPrep™ (a non-ionic solution of 60% iodixanol) here, which has proved to be biocompatible, has low osmotic pressure and low intrinsic viscosity, and suitable for the culture of multiple types of cells in microdroplets (Brower et al, 2020;Hsu et al, 2018). Researchers have used 50% OptiPrep™ to isolate quiescent yeast cells, and the results show that the isolated cells have growth behaviour similar to untreated cells and remain resist to heat shock and zymolyase treatment (Quasem et al, 2017).…”

Section: The Growth Of Single P Pastoris Cells In Picoliter Microdmentioning

confidence: 99%

Microdroplet enabled cultivation of single yeast cells correlates with bulk growth and reveals subpopulation phenomena

Liu

Peng

et al. 2020

Biotech & Bioengineering

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Yeast has been engineered for cost‐effective organic acid production through metabolic engineering and synthetic biology techniques. However, cell growth assays in these processes were performed in bulk at the population level, thus obscuring the dynamics of rare single cells exhibiting beneficial traits. Here, we introduce the use of monodisperse picolitre droplets as bioreactors to cultivate yeast at the single‐cell level. We investigated the effect of acid stress on growth and the effect of potassium ions on propionic acid tolerance for single yeast cells of different species, genotypes, and phenotypes. The results showed that the average growth of single yeast cells in microdroplets experiences the same trend to those of yeast populations grown in bulk, and microdroplet compartments do not significantly affect cell viability. This approach offers the prospect of detecting cell‐to‐cell variations in growth and physiology and is expected to be applied for the engineering of yeast to produce value‐added bioproducts.

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“…This shift in trend is due to the better physiological resemblance of iodixanol solutions, leading to better preservation of cell organelles during the isolation step. Iodixanol-based density gradients have also been proven to be a better candidate than Ficoll-based density gradients (Mita et al, 2010; Quasem et al, 2017). For example, Quasem and colleagues showed the loss of “quiescence” in yeast cells when centrifuged with Ficoll-based density gradients compared to the former.…”

Section: Resultsmentioning

confidence: 99%

Isolation of nuclei and downstream processing of cell-type-specific nuclei from micro-dissected mouse brain regions – techniques and caveats

Chongtham

Todorov

Wettschereck

et al. 2020

Preprint

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The mammalian brain consists of several structurally and functionally distinct regions equipped with an equally complex cell-type system. Due to its relevance in uncovering disease mechanisms, the study of cell-type-specific molecular signatures of different brain regions has increased. The rapid evolution of newer and cheaper sequencing techniques has also boosted the interest in cell-type-specific epigenetic studies. In fact, the nucleus holds most of the cell’s epigenetic information and is quite resistant to tissue dissociation processes as compared to cells. As such, nuclei are continually preferred over cells for epigenetic studies. However, the isolation of nuclei from cells is still a biochemically complex process, with every step affecting downstream results. Therefore, it is necessary to use protocols that fit the experimental design to yield nuclei of high quality and quantity. However, the current protocols are not suitable for nuclei isolation of small volumes of micro-dissected brain regions from individual mouse brains.Additionally, the caveats associated with centrifugation steps of nuclei extraction and the effects of different buffers have not been thoroughly investigated. Therefore, in this study, we describe an iodixanol based density gradient ultracentrifugation protocol suitable for micro-dissected brain regions from individual mice using ArccreERT2 (TG/WT).R26CAG-Sun1-sfGFP-Myc (M/WT or M/M). This mouse model shows sfGFP expression (sfGFP+) in the nuclear membrane of specific stimulus activated cells, thereby providing a good basis for the study - nuclei isolation and separation of cell-type-specific nuclei. The study also introduces new tools for rapid visualization and assessment of quality and quantity of nascent extracted nuclei. These tools were then used to examine critical morphological features of nuclei derived from different centrifugation methods and the use of different buffers to uncover underlying effects. Finally, to obtain cell-type-specific nuclei (sfGFP+ nuclei) from the isolated nuclei pool of high viscosity, an optimized protocol for fluorescence activated nuclei sorting (FANS) was established to speed up sorting. Additionally, we present a 1% PFA protocol for fixation of isolated nuclei for long term microscopic visualization.

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Density Separation of Quiescent Yeast Using Iodixanol

Cited by 9 publications

References 20 publications

Cellular quiescence in budding yeast

Cellular quiescence in budding yeast

Microdroplet enabled cultivation of single yeast cells correlates with bulk growth and reveals subpopulation phenomena

Isolation of nuclei and downstream processing of cell-type-specific nuclei from micro-dissected mouse brain regions – techniques and caveats

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