(A) Three populations were compared. (1) Unsynchronized: cells that had been passaged without earlier synchronization were passed through the FACS machine and diluted twice every 24 hr, with intervals of 10 and 14 hr between the two dilutions, but all cells that passed through the FACS machine were collected, regardless of their fluorescence level and then diluted. (2) Synchronized: a population was synchronized by selection for 3 days with selection at alternating 10 and 14 hr intervals. The culture was synchronized by FACS for another 3 days, by FACS selection and dilution twice every 24 hr, with intervals of 10 and 14 hr between the two dilutions. (3) Free run: a population was synchronized by selection for 3 days at 10:14 hr intervals. After 3 days of synchronization (marked by a dotted line), this culture was not subject to FACs but was simply diluted twice every 24 hr, with intervals of 10 and 14 hr between the two dilutions for an additional 3 days. All populations were diluted to ensure the population always reached the same final, pre-dilution density of ∼ 3 × 105 cells/ml, well below the density that corresponds to the end of mid-log phase growth for budding yeast (3 × 107 cells/ml). The initial, synchronized population used to start the free run was the same one that was used to start the population with continuing synchronization (Synch). The measured variable, F75/F25 is based on the distribution of fluorescence and pulse widths at each time point. For both measurements, the lowest value is set to 0% and the highest to 100%, with values scaled linearly in between and the number of events that lie between 0 and 25% for both fluorescence intensity and pulse width is counted as F25, and the number of events that lie between 75 and 100% for both fluorescence intensity and pulse width is counted as F75, and we plot log(F75/F25). (B) Schematic of the protocol for analyzing individual lineages. A single cell is deposited in a microtiter well, and its progeny are observed every 3.5 hr for 24 hr, before taking 3 objects randomly (single cells or cell clumps), and depositing them in fresh wells, observing for 24 hr, and finally taking 3 objects randomly (cells or cell clumps), from each of the three wells and depositing them in fresh wells, and observing for 24 hr. (C) Individual traces of lineages originating from three separate cells (Lineage 1, 2, 3). An unsynchronized population was used to establish lineages that arose from single cells, which were diluted once every 24 hr and never experienced any FACS synchronization. A single cell was placed in 100 µl of medium in a microtiter well and allowed to proliferate for 24 hr that corresponds to a maximum of 16 divisions, a 64,000-fold increase in cell density from 10 cells/ml to 640,000 cells/ml, more than 40-fold below the density at which the exponential rate of cell proliferation starts to fall. Individual cells and clumps were imaged every 3.5 hr for 72 hr. At each time point, at least 50% of the cells and clumps in the wells were imaged and the average two-dimensional area was calculated. All lineages produce approximately 24-hr oscillations without any FACS-based synchronization. Some lineages alter their phase but continue to produce ∼24-hr oscillations.