A Multiparameter Flow Cytometric Study of the Cell Cycle Based on Nuclear DNA and Protein
Markel, David C.
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This work concerns the development of a technique for the isolation of human peripheral blood lymphocytic nuclei and the subsequent staining of nuclear DNA and protein. The methodology is closely related to that of A. Pollack et. al whose protocol proved itself unreproducible on the Coulter Epics C flow cytometer and the appropriate software. The modified technique enables the exploration of several aspects of cellular kinetics using flow cytometry and computer software. The approach permits the isolation and identification of seven separate cell cycle compartments: GQ, GIA, G18, Early S, Late S, G2, and M. This work represents the following modifications of the prescribed method: use and development of a modified nuclear isolation buffer (NIB), the use and modification of several staining reagents, the use of a new filtering system, and different filter specifications, and application of the technique on a prototype Coulter• Epics C flow cytometer and software developed at Coulter Corporation and the University of Michigan. The modifications of Pollack et. al. have allowed the staining time of the nuclei to be maintained at a reduced time of thirty minutes and have permitted application to the Epics C flow cytometer and newly developed software. With this capability, the seven cell cycle compartments can be delineated more quickly and clearly than in the past.Missing pages 8-11.