Wide confocal cytometry: a new approach to study proteomic and structural changes in the cell nucleus during the cell cycle.
Wide-confocal-cytometry (WCC) is a new method developed in this paper that uses a standard confocal system to gather quantitative information on contents and fine structural details of cells. The system is operated under conditions of non-confocality, in order to capture the maximum amount of light emitted by the specimen (comparable to LSC). After analysis of macromolecule content (DNA, RNA, specific proteins, lipids, etc.), cells can be sampled using conventional confocal microscopy. We analyzed the illumination and acquiring capabilities of WCC. The quantitative power of WCC was validated by analysis of cell cycle stage in Hela cells, looking at DNA content and markers for S phase and mitosis. As an example of the potential of this methodology we have documented changes in cell nucleus during the cell cycle. After mitosis the cell nucleus changes its shape from elongated to ellipsoid and remains constant until G2. This change is associated with nuclear volume increase. As nuclear volume increases, chromatin becomes decondensed in an isometric manner, probably due to the increase in gene expression and factors necessary for RNA metabolism.
Iborra
Francisco J
FJ
MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DS, UK. francisco.iborra@imm.ox.ac.uk
Buckle
Veronica
V
D002453:Cell Cycle / Q000502:physiology*; D002467:Cell Nucleus; D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods*; D040901:Proteomics / Q000379:methods*