title: Systematic characterization of cell cycle phase-dependent protein dynamics and pathway activities by high-content microscopy-assisted cell cycle phenotyping.
abstract: Cell cycle progression is coordinated with metabolism, signaling and other complex cellular functions. The investigation of cellular processes in a cell cycle stage-dependent manner is often the subject of modern molecular and cell biological research. Cell cycle synchronization and immunostaining of cell cycle markers facilitate such analysis, but are limited in use due to unphysiological experimental stress, cell type dependence and often low flexibility. Here, we describe high-content microscopy-assisted cell cycle phenotyping (hiMAC), which integrates high-resolution cell cycle profiling of asynchronous cell populations with immunofluorescence microscopy. hiMAC is compatible with cell types from any species and allows for statistically powerful, unbiased, simultaneous analysis of protein interactions, modifications and subcellular localization at all cell cycle stages within a single sample. For illustration, we provide a hiMAC analysis pipeline tailored to study DNA damage response and genomic instability using a 3-4-day protocol, which can be adjusted to any other cell cycle stage-dependent analysis.
authors.affiliation: Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), 07745 Jena, Germany.
authors.forename: Christopher
authors.initials: C
authors.lastname: Bruhn
authors.affiliation: Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), 07745 Jena, Germany.
authors.forename: Torsten
authors.initials: T
authors.lastname: Kroll
authors.affiliation: Leibniz Institute for Age Research - Fritz Lipmann Institute (FLI), 07745 Jena, Germany; Faculty of Biology and Pharmacy, Friedrich Schiller University of Jena, 07745 Jena, Germany. Electronic address: zqwang@fli-leibniz.de.
authors.forename: Zhao-Qi
authors.initials: ZQ
authors.lastname: Wang
mesh_terms: D000818:Animals; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D010641:Phenotype; D015398:Signal Transduction
chemical_list: D018797:Cell Cycle Proteins
keywords: Cell cycle profiling; DNA damage markers; Imaging; Non-invasive cell cycle assay; hiMAC