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Related Experiment Videos

Flow cytometric cell division tracking using nuclei.

J Hasbold1, P D Hodgkin

  • 1Immune Regulation Group, Medical Foundation of the University of Sydney, Centenary Institute of Cancer Medicine and Cell Biology, Newtown, Sydney, Australia. Hasbold@centenary.usyd.edu.au

Cytometry
|July 6, 2000
PubMed
Summary
This summary is machine-generated.

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Cell division history, tracked by 5-(and-6) carboxyfluorescein diacetate succinimidyl ester (CFSE) in cells, is retained in isolated nuclei. This enables detailed analysis of nuclear components during cell division using flow cytometry.

Area of Science:

  • Cell Biology
  • Immunology
  • Biochemistry

Background:

  • 5-(and-6) carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling enables cell division history tracking via flow cytometry.
  • The retention of CFSE dye in isolated nuclei for division tracking was previously unknown.
  • Successful division tracking in nuclei could facilitate new methods for analyzing nuclear component changes across cell divisions.

Purpose of the Study:

  • To determine if isolated nuclei from CFSE-labeled cells retain sufficient dye for division history analysis.
  • To explore the potential for monitoring nuclear component dynamics across cell divisions.

Main Methods:

  • Isolated nuclei from CFSE-labeled B cells using Nonidet P-40 (NP-40) lysis.
  • Fixed nuclei with paraformaldehyde and permeabilized with Tween 20 for intranuclear staining.

Related Experiment Videos

  • Simultaneously stained for bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA), and 7-aminoactinomycin D (7-AAD).

    • Main Results:

    • Purified nuclei exhibited an asynchronous cell division profile comparable to intact cells.
    • Demonstrated simultaneous tracking of cell division history and intranuclear staining (BrdU, PCNA).
    • Confirmed that cell cycle stage and division history can be concurrently determined using 7-AAD staining.

    Conclusions:

    • Cell division history is preserved in purified nuclei post-CFSE labeling.
    • Combined CFSE, intranuclear immunofluorescence, and DNA staining offers comprehensive nuclear analysis by flow cytometry.
    • This method aids in assessing nuclear component translocation and accumulation during cell division and cell cycle progression.