Flow cytometric cell division tracking using nuclei

Background: Labeling cells with 5-(and-6) carboxyfluorescein diacetate succ inimidyl ester (CFSE) allows their subsequent division history to be determ ined by flow cytometry. Whether nuclei isolated from CFSE-labeled cells ret ain any or sufficient dye to reveal the same division history was unknown. If division tracking in nuclei were possible, it would enable the developme nt 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 cel ls with nonionic detergent Nonidet P-40 (NP-40). The purified nuclei were s ubsequently fixed with paraformaldehyde and permeabilized with Tween 20 in order to perform intranuclear staining. Results: Purified nuclei displayed the equivalent asynchronous cell divisio n profile as intact cells. Furthermore, the possibility of simultaneously m onitoring division history with intranuclear staining was established by la beling bromodeoxyuridine (BrdU) incorporated into DNA during a brief pulse prior to harvesting cells. This result was verified with the staining of pr oliferating 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 call be used in combinatio n with intranuclear immunofluorescent labeling and DNA staining to provide a comprehensive analysis of nuclei by flow cytometry. This method should pr ove useful for assessing differential nuclear translocation and accumulatio n of molecular components during consecutive division rounds and during dif ferent stages of the cell cycle. (C) 2000 Wiley-Liss, Inc.