title: Preparation of Drosophila tissue culture cells from different stages of the cell cycle for chromatin immunoprecipitation using centrifugal counterflow elutriation and fluorescence-activated cell sorting.
abstract: Many nuclear proteins alter their localization during the cell cycle. This includes proteins which regulate and execute cell cycle events and proteins involved in transcription and DNA repair. The core components of chromatin, the histone proteins, also change their modification state through the cell cycle. Chromatin immunoprecipitation (ChIP) makes it possible to localize chromatin-associated proteins to specific sequences in the genome and has revolutionized studies of transcription. Fewer studies have used ChIP to analyze protein localization or modification at specific stages in the cell cycle. This is in part because these studies require isolation of pure populations of cells at each stage of the cell cycle, which is challenging for many cell types. However, the ability to carry out ChIP from cells at specific stages in the cell cycle in some systems has revealed cell cycle regulation of chromatin localization, and cell cycle stage-specific functions and modification of chromatin proteins, providing incentive to pursue these experiments. This chapter presents protocols for isolating Drosophila S2 cells from all phases of the cell cycle using centrifugal elutriation and fluorescent-activated cell sorting. These cells are suitable for ChIP analysis.
authors.affiliation: Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: Nicole E
authors.initials: NE
authors.lastname: Follmer
authors.forename: Nicole J
authors.initials: NJ
authors.lastname: Francis
mesh_terms: D000818:Animals; D002453:Cell Cycle; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D002498:Centrifugation / Q000379:methods; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D059006:Chromosomes, Insect / Q000187:drug effects / Q000378:metabolism; D003078:Colchicine / Q000494:pharmacology; D004330:Drosophila / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D008940:Mitotic Index; D014158:Transcription, Genetic
chemical_list: D002843:Chromatin; D003078:Colchicine