ArticleNature protocols2013
Merging high-quality biochemical fractionation with a refined flow cytometry approach to monitor nucleocytoplasmic protein expression throughout the unperturbed mammalian cell cycle.
Article in Nature protocols, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 62 papers.
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Who cites it
62 citing papers in PubMed, 133 citations in OpenAlex.
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- Design, synthesis, and biological evaluation of pyrazole-linked aloe emodin derivatives as potential anticancer agents.RSC medicinal chemistry · 2021Article
- K-means quantization for a web-based open-source flow cytometry analysis platform.Scientific reports · 2021Article
- Targeting DNA Homologous Repair Proficiency With Concomitant Topoisomerase II and c-Abl Inhibition.Frontiers in oncology · 2021Article
- HStem cell research & therapy · 2020Article
- Revealing Dynamic Protein Acetylation across Subcellular Compartments.Journal of proteome research · 2020Article
- Integrative functional genomics decodes herpes simplex virus 1.Nature communications · 2020Article
2 more citing papers are in PubMed but not listed here.
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
This protocol describes a method for nucleocytoplasmic protein tracking during normal cell cycle progression using unmanipulated, asynchronous cells. In contrast with prevalent traditional methods, our approach does not require time-consuming, perturbing cell synchronization or separation. To this end, we chose a single-cell approach and developed a flow cytometry assay that is applied to whole cells and isolated nuclei. Our protocol involves a stepwise biochemical fractionation procedure to purify nuclei from whole cells, conventional DNA and indirect immunostaining techniques for the dual labeling of cells and nuclei for DNA and protein, and a refined concept of flow cytometric data processing and calculation: through the specific combination of DNA and cell size analyses, G1, S and G2/M phases of the cell cycle are further dissected to establish a high-resolution map of cell cycle progression, to which protein expression in cells or nuclei is correlated. In a final data analysis step, cell cycle-related, cytoplasmic protein expression is calculated on the basis of results obtained for whole cells and isolated nuclei. A minimum of 8 h is required to complete the procedure. As the approach does not require cell type-restricting pretreatments, numerous cell types of different origin can be readily studied. Human amniotic fluid stem cells, primary human fibroblasts, immortalized mouse fibroblasts and transformed tumor cells are analyzed at comparable efficiencies, demonstrating low intercell assay variability.
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