Automated Cell Cycle Analysis Figure 5. TSA detects decreases in mitotic duration when the spindle assembly checkpoint is inactivated. HeLa H2B-GFP cells were untransfected or transfected with non-targeting control, MAD2 or BubR1 siRNAs and were imaged 24 hours post-transfection, every 4 min for 48 hours. For each condition, 3 fields in 2 independent wells were imaged and the results for the 6 image series were combined. Image series were analyzed using DCELLIQ and half of the movies were also analyzed manually. Cumulative frequency curves for the cell populations are provided. doi:10.1371/journal.pone.0025511.g005 in determining the duration of interphase under conditions that permit cells to divide at least twice in a movie. We also used TSA to measure how cycloheximide treatment affects mitotic duration. Interestingly, whereas low dose (0.025– 0.1 mg/mL) cycloheximide increased interphase duration, it significantly shortened mitotic duration (Figure 6b; p# 2.6561027). At a higher concentration (1 mg/mL), cycloheximide significantly prolonged mitosis (p = 1.8261026). These findings highlight the utility of the TSA to identify effects that prolong one cell cycle phase while shortening another. As an alternative method to increase interphase duration, we treated HeLa H2B-GFP cells with doxorubicin, a topoisomerase II inhibitor which induces formation of double strand breaks and G2 phase cell cycle arrest in HeLa cells [19]. We used concentrations between 10 and 100 nM to identify drug concentrations that would delay interphase progression but still permit mitotic entry (Figure 7a). The time-series approach detected a one-hour increase in median interphase duration in cells treated with 10 nM doxorubicin, compared to DMSO treated cells (p = 0.00008). At higher dose (25 nM), median duration increased further to 25.4 hours. At this concentration, the number of measured interphase events decreased as fewer cells entered mitosis. At concentrations greater than 25 nM, fewer mitotic events could be observed and interphase duration could not be measured. We also measured mitotic duration in these cells. Treatment with 10– 25 nM doxorubicin induced a statistically significant and dose- dependent increase in mitotic duration (Figure 7b) compared to DMSO-treated samples (p#4.2861029). In conclusion, TSA enabled identification of statistically significant changes in both interphase and mitotic duration upon multiple chemical treat- ments. Discussion Here we describe the development of a time-series based approach for automatic identification of mitotic and interphase duration from time-lapse movies of dividing cells. We show that this method can detect small changes in interphase or mitotic duration, with sensitivity comparable to changes that can be detected by manual analysis. We show that the approach can be used on a second cell line by adjusting the frequency of image acquisition to take in account differences in baseline mitotic duration. Compared to SVM-based approaches, the time-series approach is more tolerant of changing experimental conditions, does not require extensive training, and is computationally rapid. Only two features, area and average intensity, are required to accurately identify the transition points between interphase and mitosis. These features can be easily calculated from images of relatively low magnification (206) wide-field fluorescent imaging. The DCELLIQ platform is self-sufficient, making it possible to analyze large data sets with little need for attendance by the user. A fundamental requirement of our method is that the imaging frequency must be sufficient to capture at least three images during PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e25511