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To accurately observe high-frequency events during transient cell cycle kinetics, we have implemented a single step 15-min DNA staining protocol using automated flow cytometry. This protocol was used to sample and to analyze a Chinese hamster ovary cell culture for the DNA distribution, viable cell concentration, apoptotic cell concentration, and light scattering properties every 25 min over 4.5 days in response to a nutrient deprivation and a nutrient upshift. After the nutrient deprivation and exposure to fresh growth medium, two populations of cells started proliferating at different times likely corresponding to cells leaving the G0 and G1 cell cycle phases. After a nutrient upshift in late exponential growth, a cell cycle arrest occurred at the G1/S and G2/M boundary. The resulting cell cycle and proliferation kinetics followed damped oscillations that directly reveal the average time cells spend in each cell cycle phase. The observed detailed dynamics of the cell cycle progression is made possible through the high-frequency sampling enabled by automated flow cytometry. The approach should be useful in studying cell cycle perturbations in response to different environmental conditions resulting from exposure to specific nutrients or to drugs. ' 2008 International Society for Advancement of CytometryKey terms automated flow cytometry; population dynamics; cell cycle staining; digitonin; mammalian cell culture; single cell heterogeneity DETERMINATIONof the cell cycle distribution is one of the most basic and most widely used applications of flow cytometry in basic biological research and in diagnostic applications. It traditionally involves cell fixation and permeabilization and cell staining with nucleic acid specific dyes, often after enzymatic removal of RNA. Variations of this methodology have been well documented in many reports, review articles and textbooks (1). The cell preparation and staining steps typically involve several manual operations, and their careful execution determines the reproducibility and the quality of the cell cycle distribution that can be obtained. However, in many cases a large fraction of cells may be lost during fixation and staining steps which introduces the additional problem that the remaining cells may not be representative of the entire sample.Traditional cell cycle staining protocols use either cell membrane permeable or impermeable dyes. Staining protocols using membrane permeable dyes are typically simple and short involving only addition of the dye to the cells. However, membrane permeable dyes often are more expensive in comparison to membrane impermeable dyes [Vybrant, DRAQ5 (2)] or limited to a UV excitation source (Hoechst, DAPI). In contrast, membrane impermeable dyes such as PI or Acridine Orange are inexpensive and can be excited by common 488 nm light sources. Cell cycle staining protocols using membrane impermeable dyes are usually long, multistep procedures.Time dependent changes in the cell cycle distribution of a growing cell population reflect t...

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We have developed an instrument based on a flow cytometer platform that is capable of tracking individual, suspended cells over extended time periods. The instrument repeatedly moves in a capillary the same volume segment of fluid containing tens to hundreds of suspended cells through the focal point of a laser. Individual cells are then tracked based on the timing of when they cross the laser, and cell properties are measured as in a conventional flow cytometer. Because cells are repeatedly measured the single-cell rates of change can be determined. The developed instrumentation was applied to measure the variability of ABC transporter activity in a population of human cancer cells and the temperature dependence of constitutively expressed Gfp in yeast. A wide range of transport rates can be observed in the cancer cell population while the single-cell Gfp fluorescence in yeast shows pronounced oscillations in response to temperature shifts. These observations are not detectable at the population level. Therefore, such measurements are useful for investigating cell function as they reveal how variable properties of single cells change over time. ' 2010 International Society for Advancement of Cytometry Key termssingle cell dynamics; rates of change; drug resistance IN both medicine and biotechnology, understanding how variability of properties arises and changes over time is of fundamental importance. Variability of properties has been indicated in the progression of certain diseases (1) and in explaining the productivity of chemicals or pharmaceuticals with engineered cultures (2,3). This variability has been generally related to genetic differences, to different positions in the cell cycle, to the exposure of a heterogeneous environment, or to stochastic variations due to the low number of molecules in individual cells. While the variability in the single-cell composition can be readily evaluated using conventional flow cytometry, the variability in dynamic properties of cells that reflect the rate at which cells change, are rather difficult to obtain and few examples exist in the literature.To experimentally measure the rate of how properties change, one must measure a property using either an Eulerian or Lagrangian reference frame (4,5). In the Eulerian reference frame one measures the distribution of a property in a cell population at discrete time points. In contrast, in the Lagrangian reference frame one follows individual cells over time and observes how their properties change. The properties of the entire cell population are then obtained as the sum of contributions of the individually tracked cells. The two reference frames are analogous to the fluid mechanic problem of converting Eulerian observations to Lagrangian (4).In the Eulerian reference frame, flow cytometry is primarily used to generate experimental data. This instrument yields a snapshot of the cellular property distribution at different time points and cells are discarded after the measurement. The ability to accurately understand...