title: Isolation of Plant Nuclei at Defined Cell Cycle Stages Using EdU Labeling and Flow Cytometry.
abstract: 5-Ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that can be rapidly incorporated into replicating DNA in vivo and, subsequently, detected by using "click" chemistry to couple its terminal alkyne group to fluorescent azides such as Alexa Fluor 488. Recently, EdU incorporation followed by coupling with a fluorophore has been used to visualize newly synthesized DNA in a wide range of plant species. One particularly useful application is in flow cytometry, where two-parameter sorting can be employed to analyze different phases of the cell cycle, as defined both by total DNA content and the amount of EdU pulse-labeled DNA. This approach allows analysis of the cell cycle without the need for synchronous cell populations, which can be difficult to obtain in many plant systems. The approach presented here, which was developed for fixed, EdU-labeled nuclei, can be used to prepare analytical profiles as well as to make highly purified preparations of G1, S, or G2/M phase nuclei for molecular or biochemical analysis. We present protocols for EdU pulse labeling, tissue fixation and harvesting, nuclei preparation, and flow sorting. Although developed for Arabidopsis suspension cells and maize root tips, these protocols should be modifiable to many other plant systems.
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 27695, USA.
authors.forename: Emily E
authors.initials: EE
authors.lastname: Wear
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695, USA.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Concia
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695, USA.
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Brooks
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 27695, USA.
authors.forename: Emily A
authors.initials: EA
authors.lastname: Markham
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 27695, USA.|Syngenta Crop Protection, LLC, Research Triangle Park, NC, 27709, USA.
authors.forename: Tae-Jin
authors.initials: TJ
authors.lastname: Lee
authors.affiliation: Department of Horticultural Science, North Carolina State University, Raleigh, NC, 27695, USA.
authors.forename: George C
authors.initials: GC
authors.lastname: Allen
authors.affiliation: Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC, 27695, USA.
authors.forename: William F
authors.initials: WF
authors.lastname: Thompson
authors.affiliation: Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC, 27695, USA. linda_hanley-bowdoin@ncsu.edu.
authors.forename: Linda
authors.initials: L
authors.lastname: Hanley-Bowdoin
mesh_terms: D017360:Arabidopsis / Q000166:cytology* / Q000235:genetics*; D002453:Cell Cycle; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus / Q000235:genetics*; D057930:Click Chemistry / Q000379:methods; D004261:DNA Replication; D018744:DNA, Plant / Q000032:analysis / Q000235:genetics; D003857:Deoxyuridine / Q000031:analogs & derivatives / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D003313:Zea mays / Q000166:cytology* / Q000235:genetics*
chemical_list: D018744:DNA, Plant; D005456:Fluorescent Dyes; C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine
keywords: 5-Ethynyl-2′-deoxyuridine; Arabidopsis; Cell cycle; Chromatin; DNA replication; Flow cytometry; Maize; Nuclei sorting; Percoll gradient