Cell Cycle Analysis Using In Vivo Staining of DNA-Synthesizing Cells

Methods Mol Biol. 2020:2150:141-152. doi: 10.1007/7651_2019_228.

Abstract

The thymidine analogues BrdU (5-bromo-2´-deoxyuridine) and EdU (5-ethynyl-2´-deoxyuridine) are routinely used for determination of the cells synthesizing DNA in the S-phase of the cell cycle. Availability of the anti-BrdU antibody clone MoBu-1 detecting only BrdU allowed to develop a method for the sequential DNA labelling by these two thymidine analogues for determining the cell cycle kinetic parameters.In the current step-by-step protocol, we present` two approaches optimized for in vivo study of the cell cycle and the limitations that such approaches imply: (1) determination of the cell flow rate into the G2-phase by dual EdU/BrdU DNA-labelling method and (2) determination of the outflow of DNA-labelled cells arising from the mitosis.

Keywords: Antibodies; Cell cycle; DNA labellin`g; Flow cytometry; Immunophenotyping; S-phase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bone Marrow Cells / metabolism
  • Bromodeoxyuridine / metabolism
  • Cell Cycle*
  • Cell Differentiation
  • DNA / biosynthesis*
  • Data Analysis
  • Deoxyuridine / analogs & derivatives
  • Flow Cytometry
  • Immunophenotyping
  • Mice
  • Mitosis
  • Rheology
  • S Phase
  • Staining and Labeling / methods*

Substances

  • DNA
  • Bromodeoxyuridine
  • 5-ethynyl-2'-deoxyuridine
  • Deoxyuridine