Automated Cell Cycle Analysis Figure 2. The choice of imaging frequency influences measurement of mitotic duration. HeLa H2B-GFP cells were imaged every 4 minutes for 24 hours and all images (4 min interval) or selected images (8 or 12 min intervals) were analyzed with the automated approach. For each condition 4 fields in 2 independent wells were imaged and the results for the 8 images series were combined. Half of the movies were analyzed manually for comparison. Cumulative frequency curves of events are depicted as a function of mitotic duration for the different intervals. The number of detected mitotic events (N), measured median and mean mitotic duration are provided. doi:10.1371/journal.pone.0025511.g002 interval (12 minutes). In analyzing the underlying reason for the bias, we observed that some cells that were not tracked moved on top of adjacent interphase or mitotic cells after entering mitosis, causing the two cell nuclei to overlap or completely cross each other. This results in segmentation difficulties and makes proper tracking nearly impossible, leading to exclusion of these cells from analysis by the trace removal feature of the program. Interestingly, these cells were found to spend a longer time in mitosis, possibly because their detachment perturbs spindle formation or mitotic regulation. We compared the average duration measured by the TSA (N = 43) with the average determined from manual analysis of all cells in the movie either tracked or not tracked (N = 112), and found that the underestimation in average mitotic duration was not statistically significant for this small population (p = 0.0915). However, such a trend becomes statistically significant when a larger number of cells are analyzed (Figure 3b). We next determined whether the TSA can successfully identify small increases in mitotic duration. We treated HeLa H2B-GFP cells with DMSO or low doses of nocodazole, which prolongs mitosis by activating the spindle checkpoint [16,17]. We analyzed 9 movies per condition by TSA and manually analyzed 4 of these movies to provide sufficient data for comparison (Figure 3b). In this experiment, the manual analysis included all cells that were imaged, so that the effects of the selection bias could be evaluated. The TSA detected a statistically significant increase in mitotic duration from an average of 69.5 min for DMSO to 73.8 min for 5 nM nocodazole treatment (p = 0.0028). The TSA identified the increase in average mitotic duration with high statistical significance (p = 5.06610211) upon treatment with a higher dose of nocodazole (10 nM). For each condition, the TSA slightly under-measured average mitotic duration as compared to manual analysis, consistent with the bias described above. However, the selection bias did not prevent the program from detecting the increase in average mitotic duration at higher drug concentrations (15–50 nM nocodazole; Figure S2). Despite a slight under-measurement as a consequence of the selection bias, these experiments indicate that the TSA can detect subtle increases in average mitotic duration, and can accurately determine median mitotic duration. We next determined whether the same algorithm could be used to analyze changes in mitotic duration in a different cell line (A549 H2B-GFP cells, a lung cancer cell line), which has a shorter average mitotic duration (30 minutes) compared to our HeLa H2B-GFP cells. In order for DCELLIQ to accurately identify mitotic transitions, we acquired images every 5 minutes, but did not otherwise alter any image analysis parameters. As for HeLa cells, the TSA detected an increase in mitotic duration upon nocodazole treatment (Figure 4). For control-treated A549 H2B- GFP cells, the time-series method accurately measured average mitotic duration as 30 minutes. Treatment with 15 nM nocoda- zole caused no significant change in mitotic duration but 25 nM nocodazole resulted in a significant increase in average mitotic duration to 34.8 and 37.8 min as measured by automated (p = 0.00043) and manual (p = 2.14610212) analysis, respectively. These results indicate that the time-series method can be used to detect changes in duration of mitosis in another cell line if the image sampling frequency is adjusted appropriately. TSA can identify decreases in mitotic duration To determine whether the time-series method can detect experimental perturbations that shorten mitotic duration, we analyzed mitotic timing in cells in which the spindle assembly checkpoint was inactivated by RNAi (Figure 5). HeLa H2B-GFP cells were depleted of the proteins MAD2 or BubR1 by siRNA- based transfection, which is known to shorten mitotic duration [4]. PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e25511