Cytometry and cell biology; The bright side of errors

Optical flow cytometry is widely used for isolating cells of interest from a heterogeneous cell population. Applications of this technology in cell cycle research are typically limited to DNA and DNA replication quantification, as well as to other fluorescence-based profiling of cell cycle progression. Size is a basic characteristic of every cell, with proliferating cells being no different. This metric, however, cannot be measured directly by standard cell sorters. Consequently, applications of cytometry in cell size research are underused, and often misused. We demonstrated how to properly approximate cell size by light scatter parameters available in all standard cytometers. These protocols enabled us to accurately sort cells by size, as well as to synchronize them in the G1 phase without cell cycle blocking agents or chemical labeling. Furthermore, by integrating information on the cell cycle and using different instrument configurations, we extended the use of size-based analysis and sorting for purifying early and late cytokinetic cells directly from an asynchronous population of normally proliferating cells. We show valuable applications of these new methodologies in studying cell cycle, cell size, and their interrelationships.