Timing the changes of cyclin E cell content in G1 in exponentially growing cells

Daniela Tomasoni, Monica Lupi, Fadia Bekkal Brikci, Paolo Ubezio

Research output: Contribution to journalArticlepeer-review

Abstract

We present a method for measuring the content of immunocytochemically detected proteins in individual cells progressing through G1 phase and its application in the analysis of cyclin E levels. The sequence of G1 events is tracked under unaltered cycling conditions, in a cell line in the phase of balanced growth in vitro, to avoid the pitfalls of synchronization. Cells were pulse-labeled with BrdUrd and analyzed sequentially by multiparameter flow cytometry, focusing on the subpopulation of labeled cells progressively entering G1. We use the time-from-birth ("age") of individual cells to track their position inside G1. Using the average content of cyclin E in the whole population of G1 cells as the internal reference for each sample, we analyzed the time course of the frequency histograms of cyclin E content within BrdUrd-labeled G1 cells by exploiting the properties of the age distributions of asynchronous populations. This way we could calculate the average cyclin E content of cells in each age cohort. Cyclin E values were low until age 3 h, after which they rose gradually, reaching six times the value of newborn cells at the end of G1.

Original languageEnglish
Pages (from-to)158-167
Number of pages10
JournalExperimental Cell Research
Volume288
Issue number1
DOIs
Publication statusPublished - Aug 1 2003

Keywords

  • Cell cycle
  • Cell proliferation
  • Cyclins
  • Flow cytometry
  • Mathematical models

ASJC Scopus subject areas

  • Cell Biology

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