Dataset Information

Triple S-Phase Labeling of Dividing Stem Cells.

ABSTRACT: Marking replicating DNA with multiple labels presents the possibility of revealing new features and mechanisms of DNA synthesis and cell division; however, progression beyond double labeling has been hampered by cross-reactivity of label detection and scarcity of appropriate labels. Here, we present a method for triple S-phase labeling of the dividing cells, with a fourth label used to mark cells actively engaged in cell-cycle progression (e.g., using Ki67) or to phenotype the dividing cells or their progeny (e.g., using a GFP-expressing lineage reporter transgene). We apply this method to determine the parameters of neural stem cell division in the adult brain, to birth date up to four cohorts of dividing cells, and to reveal patterns of stem cell division in non-neural tissues.

SUBMITTER: Podgorny O

PROVIDER: S-EPMC5830949 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Triple S-Phase Labeling of Dividing Stem Cells.

Podgorny Oleg O Peunova Natalia N Park June-Hee JH Enikolopov Grigori G

Stem cell reports 20180201 2

Marking replicating DNA with multiple labels presents the possibility of revealing new features and mechanisms of DNA synthesis and cell division; however, progression beyond double labeling has been hampered by cross-reactivity of label detection and scarcity of appropriate labels. Here, we present a method for triple S-phase labeling of the dividing cells, with a fourth label used to mark cells actively engaged in cell-cycle progression (e.g., using Ki67) or to phenotype the dividing cells or ...[more]

PMID: 29358087

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Project description:Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

Dataset Information

Triple S-Phase Labeling of Dividing Stem Cells.

Publications

Triple S-Phase Labeling of Dividing Stem Cells.

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