J Hasbold1, P D Hodgkin. 1. Immune 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
Abstract
BACKGROUND: Labeling cells with 5-(and-6) carboxyfluorescein diacetate succinimidyl ester (CFSE) allows their subsequent division history to be determined by flow cytometry. Whether nuclei isolated from CFSE-labeled cells retain any or sufficient dye to reveal the same division history was unknown. If division tracking in nuclei were possible, it would enable the development of new methods for monitoring quantitative changes in nuclei components and how these might vary with successive divisions. METHODS: Nuclei from CFSE-labeled B cells were prepared by lysing whole cells with nonionic detergent Nonidet P-40 (NP-40). The purified nuclei were subsequently fixed with paraformaldehyde and permeabilized with Tween 20 in order to perform intranuclear staining. RESULTS: Purified nuclei displayed the equivalent asynchronous cell division profile as intact cells. Furthermore, the possibility of simultaneously monitoring division history with intranuclear staining was established by labeling bromodeoxyuridine (BrdU) incorporated into DNA during a brief pulse prior to harvesting cells. This result was verified with the staining of proliferating cell nuclear antigen (PCNA). In addition, aminoactinomycin D (7-AAD) staining established that cell cycle stage and cell division history could be simultaneously determined. CONCLUSIONS: Our results demonstrate that cell division history is retained in purified cell nuclei after CFSE labeling and can be used in combination with intranuclear immunofluorescent labeling and DNA staining to provide a comprehensive analysis of nuclei by flow cytometry. This method should prove useful for assessing differential nuclear translocation and accumulation of molecular components during consecutive division rounds and during different stages of the cell cycle. Copyright 2000 Wiley-Liss, Inc.
BACKGROUND: Labeling cells with 5-(and-6) carboxyfluorescein diacetate succinimidyl ester (CFSE) allows their subsequent division history to be determined by flow cytometry. Whether nuclei isolated from CFSE-labeled cells retain any or sufficient dye to reveal the same division history was unknown. If division tracking in nuclei were possible, it would enable the development of new methods for monitoring quantitative changes in nuclei components and how these might vary with successive divisions. METHODS: Nuclei from CFSE-labeled B cells were prepared by lysing whole cells with nonionic detergent Nonidet P-40 (NP-40). The purified nuclei were subsequently fixed with paraformaldehyde and permeabilized with Tween 20 in order to perform intranuclear staining. RESULTS: Purified nuclei displayed the equivalent asynchronous cell division profile as intact cells. Furthermore, the possibility of simultaneously monitoring division history with intranuclear staining was established by labeling bromodeoxyuridine (BrdU) incorporated into DNA during a brief pulse prior to harvesting cells. This result was verified with the staining of proliferating cell nuclear antigen (PCNA). In addition, aminoactinomycin D (7-AAD) staining established that cell cycle stage and cell division history could be simultaneously determined. CONCLUSIONS: Our results demonstrate that cell division history is retained in purified cell nuclei after CFSE labeling and can be used in combination with intranuclear immunofluorescent labeling and DNA staining to provide a comprehensive analysis of nuclei by flow cytometry. This method should prove useful for assessing differential nuclear translocation and accumulation of molecular components during consecutive division rounds and during different stages of the cell cycle. Copyright 2000 Wiley-Liss, Inc.
Authors: Svetlana N Radyuk; Igor Rebrin; James M Luchak; Katarzyna Michalak; Vladimir I Klichko; Rajindar S Sohal; William C Orr Journal: J Biol Chem Date: 2008-11-26 Impact factor: 5.157