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1.
Figure 7

Figure 7. TSA detects changes in interphase duration in response to doxorubicin treatment.. From: A Time-Series Method for Automated Measurement of Changes in Mitotic and Interphase Duration from Time-Lapse Movies.

HeLa H2B-GFP cells were treated with doxorubicin at indicated concentrations. Cells were imaged every 12 min for 48 hours. For each condition, 4 fields in 2 independent wells were imaged and the results for the 8 image series were combined. Image series were analyzed using DCellIQ with TSA. Cumulative frequency curves of interphase (a) and mitotic (b) durations for the cell populations are provided.

Frederic D. Sigoillot, et al. PLoS One. 2011;6(9):e25511.
2.
Figure 5

Figure 5. TSA detects decreases in mitotic duration when the spindle assembly checkpoint is inactivated.. From: A Time-Series Method for Automated Measurement of Changes in Mitotic and Interphase Duration from Time-Lapse Movies.

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.

Frederic D. Sigoillot, et al. PLoS One. 2011;6(9):e25511.
3.
Figure 6

Figure 6. TSA detects changes in interphase and mitotic durations in response to cycloheximide treatment.. From: A Time-Series Method for Automated Measurement of Changes in Mitotic and Interphase Duration from Time-Lapse Movies.

HeLa H2B-GFP cells were treated with cycloheximide at indicated concentrations. Cells were imaged every 12 min for 48 hours. For each condition, 4 fields in 2 independent wells were imaged and the results for the 8 image series were combined. Image series were analyzed using DCellIQ (a and b) with the time series approach and half of them were analyzed manually (a). The origin of x-axis in graph (a) was set at 800 minutes as no interphase events had a duration shorter than 800 minutes.

Frederic D. Sigoillot, et al. PLoS One. 2011;6(9):e25511.
4.
Figure 1

Figure 1. ‘Area’ and ‘Average Intensity’ features are sufficient to identify the boundaries of mitosis.. From: A Time-Series Method for Automated Measurement of Changes in Mitotic and Interphase Duration from Time-Lapse Movies.

(a) A portion of an image of HeLa cells expressing the fluorescent marker H2B-GFP is shown. The red asterisk marks an example of a nucleus automatically tracked throughout a division. Images were acquired every 12 minutes. At the time of division (between frames 4 and 5) each daughter nucleus is tracked independently to produce a “trace” for the division. The images of the trace are then used for the time-series analysis as shown in (b). (b) An example of a trace, showing the values of average intensity (I) and area (A) graphed over time with montage of nucleus images. The interphase-prophase transition (IPT) and metaphase-anaphase transition (MAT) points are identified.

Frederic D. Sigoillot, et al. PLoS One. 2011;6(9):e25511.
5.
Figure 4

Figure 4. TSA detects small increases in mitotic duration when the spindle assembly checkpoint is activated by nocodazole in another cell line.. From: A Time-Series Method for Automated Measurement of Changes in Mitotic and Interphase Duration from Time-Lapse Movies.

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.

Frederic D. Sigoillot, et al. PLoS One. 2011;6(9):e25511.
6.
Figure 2

Figure 2. The choice of imaging frequency influences measurement of mitotic duration.. From: A Time-Series Method for Automated Measurement of Changes in Mitotic and Interphase Duration from Time-Lapse Movies.

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.

Frederic D. Sigoillot, et al. PLoS One. 2011;6(9):e25511.
7.
Figure 3

Figure 3. TSA detects small increases in mitotic duration when the spindle assembly checkpoint is activated by nocodazole.. From: A Time-Series Method for Automated Measurement of Changes in Mitotic and Interphase Duration from Time-Lapse Movies.

(a) HeLa H2B-GFP cells were imaged every 12 min for 48 hours after treatment with DMSO. Mitotic events from one movie, for nuclei that were successfully tracked, were measured by TSA (tracked successfully) and duration of these events was manually determined and both measurements were compared. Mitotic duration was also manually determined for cells that were not tracked successfully. Such cells were either not present in the imaging frame at all time throughout the movie or their trace showed potential errors of segmentation or tracking and were as a result excluded by 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).

Frederic D. Sigoillot, et al. PLoS One. 2011;6(9):e25511.

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