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Histochemistry

, Volume 76, Issue 1, pp 113–121 | Cite as

Flow cytometric analysis of chromosomes and cells using a modified BrdU-hoechst method

  • E. Severin
  • B. Ohnemus
Article

Summary

Chromosomes and interphase cells were harvested from cultures of the Chinese hamster line B14 F28 grown in medium containing BrdU up to four cell cycles and stained with the fluorescent dye 33342 Hoechst for flow cytometry. The newly synthetized BrdU-DNA is not stainable by the Hoechst dye which is highly specific for thymidine. The temporal development of the DNA fluorescence after addition of BrdU to the growth medium has been investigated. The chromosomal fluorescence intensity is reduced one step per generation. The extent of the intensity decrease by BrdU incorporation is proportional to the amount of new DNA and it is realized by repeated measurement following an UV-exposure. This UV-illumination stops the quenching by BrdU of the Hoechst stain induced DNA fluorescence. Therefore, the entire DNA content of these chromosomes now becomes measurable. The obtained intensity gain serves as a measure of the extent of the previous BrdU caused intensity shift. In this way we could establish 3 successive mitoses. Principally, this method is suitable also for measurement of whole cells in order to obtain both the number of generations in the experimental period and the phase distribution of the cell cycle.

Keywords

Cell Cycle Flow Cytometry Fluorescence Intensity Growth Medium Thymidine 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag 1982

Authors and Affiliations

  • E. Severin
    • 1
  • B. Ohnemus
    • 1
  1. 1.Institut für Strahlenbiologie der Westfälischen Wilhelms-Universität MünsterMünsterGermany

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