title: Non-destructive handling of individual chromatin fibers isolated from single cells in a microfluidic device utilizing an optically driven microtool.
abstract: We report a novel method for the non-destructive handling of, and biochemical experiments with, individual intact chromatin fibers, as well as their isolation from single cells, utilizing a specifically designed microfluidic device with an optically driven microtool under the microscope. Spheroplasts of recombinant fission yeast cells expressing fluorescent protein-tagged core histones were employed, and isolation of chromatin fibers was conducted by cell bursting via changing from isotonic conditions to hypotonic conditions in the microfluidic device. The isolation of chromatin fibers was confirmed by the fluorescent protein-tagged core histones involved in the chromatin fibers. For the non-destructive handling of the isolated chromatin fibers in the microfluidic device, we developed antibody-conjugated microspheres, which had affinity to the fluorescent protein-tagged core histones, and the microspheres were manipulated using optical tweezers, which functioned as optically driven microtools. With the aid of the microtool, isolated chromatin fibers were handled non-destructively and were tethered at the microstructures fabricated in the microfluidic device with straightened conformation by the flow. Immunofluorescence staining was carried out as a demonstrative biochemical experiment with the individual native chromatin fibers isolated in the microfluidic device, and specific fluorescent spots were visualized along the tethered chromatin fibers. Thus, the potential application of this method for epigenetic analyses of intact chromatin fibers isolated from single cells is demonstrated.
authors.affiliation: Department of Mechanical Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. oana@mech.t.u-tokyo.ac.jp.
authors.forename: Hidehiro
authors.initials: H
authors.lastname: Oana
authors.forename: Kaori
authors.initials: K
authors.lastname: Nishikawa
authors.forename: Hirotada
authors.initials: H
authors.lastname: Matsuhara
authors.forename: Ayumu
authors.initials: A
authors.lastname: Yamamoto
authors.forename: Takaharu G
authors.initials: TG
authors.lastname: Yamamoto
authors.forename: Tokuko
authors.initials: T
authors.lastname: Haraguchi
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hiraoka
authors.forename: Masao
authors.initials: M
authors.lastname: Washizu
mesh_terms: D002843:Chromatin / Q000737:chemistry* / Q000302:isolation & purification*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D052898:Optical Tweezers; D012441:Saccharomyces cerevisiae / Q000166:cytology; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D002843:Chromatin