Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

Cytometry. 2000 Oct 1;41(2):89-95. doi: 10.1002/1097-0320(20001001)41:2<89::aid-cyto2>3.0.co;2-i.


Background: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells.

Methods: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry.

Results: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.

Conclusions: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.

Publication types

  • Evaluation Study

MeSH terms

  • 3T3 Cells
  • Animals
  • Antibodies, Monoclonal / immunology
  • Antimetabolites / pharmacology
  • Benzimidazoles / pharmacology*
  • Bromodeoxyuridine / immunology
  • Bromodeoxyuridine / pharmacology*
  • Cell Separation
  • Cell Survival*
  • Cells, Cultured
  • DNA / analysis
  • Flow Cytometry*
  • Fluorescent Dyes / pharmacology
  • Indicators and Reagents / pharmacology
  • Intercalating Agents / pharmacology
  • Male
  • Mice
  • Mitosis
  • Muscle, Skeletal / cytology
  • Propidium / pharmacology
  • Rats


  • Antibodies, Monoclonal
  • Antimetabolites
  • Benzimidazoles
  • Fluorescent Dyes
  • Indicators and Reagents
  • Intercalating Agents
  • Propidium
  • DNA
  • Bromodeoxyuridine
  • bisbenzimide ethoxide trihydrochloride