Automated Cell Cycle Analysis the trace removal part of the program. Sample measurements are graphed as cumulative frequency of events as a function of mitotic duration and duration means and medians are reported. (b) HeLa H2B-GFP cells treated with indicated concentrations of nocodazole or vehicle were imaged every 12 min, for 48 hours. Half of the resulting image series were analyzed manually by identifying the duration between the first frame of mitotic entry and the first frame of anaphase for all cells in the field. All image series were analyzed by DCELLIQ using TSA. For each condition, 3 fields in 3 independent wells were imaged and the results for the image series were combined. The Wilcoxon test was used to determine whether differences in mitotic duration between compared samples were statistically significant (p,0.05). doi:10.1371/journal.pone.0025511.g003 Cells were imaged for 48 hours beginning 24 hours after transfection. Images were collected every 4 minutes as cells with MAD2 or BubR1 knockdown are expected to have short mitosis in the range of 12–20 minutes [4]. For untransfected and control siRNA transfected cells, manual and TSA yielded very similar median mitotic durations of 56–58 minutes. The TSA yielded slightly shorter mean mitotic durations (57.0 and 55.4 min) compared to manual analysis (64.4 and 63.3 min) as expected based on the bias described above. In cells treated with siRNAs that target either MAD2 or BubR1, manual and time-series analysis both measured a strong reduction in median (16–20 min) and mean (23–28 min) mitotic duration (Figure 5). Interestingly, TSA did not underestimate mitotic duration in the knockdown condition, consistent with the observation that cells going through short mitosis do not round up as much and remain more attached to the culture support than cells with normal mitotic duration. Therefore, TSA can detect short mitotic events and decreases in mitotic duration, as long as cells are imaged frequently enough to acquire multiple frames during mitosis. TSA can measure changes in interphase duration Because our approach combines cell tracking over time with identification of successive mitotic divisions, it is possible to measure the duration of interphase if a cell divides at least twice during the movie. We first evaluated the effect of cycloheximide, a protein synthesis inhibitor that prevents mitotic entry at high concentrations. Low concentrations of cycloheximide (0.01 mg/ mL) decrease the rate of protein synthesis in HeLa cells by 5%, whereas high concentrations (1 mg/mL) inhibit protein synthesis by 80% [18]. We treated HeLa H2B-GFP cells with cyclohexi- mide concentrations ranging between 0.01 mg/mL and 1 mg/mL and compared the results of time-series analysis to manual analysis (Figure 6a). In untreated cells, TSA measured a median interphase duration of 20.4 h, very similar to the duration measured manually (20.6 h). At the lowest concentration of cycloheximide tested (0.01 mg/mL) TSA measured a statistically-significant increase in interphase duration (21.8 h; p = 0.00018), which was very similar to the value measured manually (21.6 h). At a higher dose of cycloheximide (0.025 mg/mL), TSA detected a further increase in interphase duration (23.8 h) that was again very similar to the value obtained manually (23.4 h). Higher concentrations of cycloheximide yielded a further increase in interphase duration (Figure S3). At 1 mg/mL, cycloheximide treatment induced a complete interphase arrest by the end of the movie resulting in no cell entering mitosis twice and thus interphase duration could not be determined. These findings indicate that TSA is highly accurate Figure 4. TSA detects small increases in mitotic duration when the spindle assembly checkpoint is activated by nocodazole in another cell line. A549 H2B-GFP cells treated with indicated concentrations of nocodazole or vehicle were imaged every 5 min for 48 hours. Half of the resulting image series were analyzed manually to identify the duration between the first frame of mitotic entry and the first frame of anaphase for all cells in the field. All image series were analyzed by DCELLIQ using the time series approach. For each condition, 4 fields in 2 independent wells were imaged and the results for the image series were combined. doi:10.1371/journal.pone.0025511.g004 PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e25511