To measure the fluorescent cyclin B1-GFP degradation in living cells, time-lapse images were collected at 1-min intervals. The region was drawn around each cell to be measured, and the identical region was placed in an area without fluorescent objects to be used for background subtraction. The net average fluorescence intensity of a pixel in the region of interest was calculated for each time point. Because cells expressed different levels of fluorescent cyclin B, the net average intensity values were normalized to the initial (first time point) value that was designated as 1. Averages of normalized intensity values of at least five identically treated cells were calculated for each time point and plotted on a graph. For these experiments, all parameters during image acquisition were the same.
To measure fluorescence intensities of MPM2, pS-Cdk, and pNucleolin antibody labeling, 1-μm Z-stacks through cells of different stages of mitosis were acquired. A region was drawn around each cell to be measured, and the same size region was drawn in an area without fluorescent objects to be used for background subtraction. The net integrated intensity for each cell was measured at a single Z plane with highest integrated intensity values in the region of interest (this was usually the plane with the best focus). The weak signal from interphase cells was designated as 1, and the fluorescence intensity values at each mitotic stage were normalized and plotted relative to interphase. Each bar represents an average of 15–30 cells. The intensity of a signal from the control slide labeled with secondary antibodies alone was comparable to the intensity of the background in experimental samples.
To measure fluorescence intensities of MPM2, pS-Cdk, and pNucleolin antibody labeling, 1-μm Z-stacks through cells of different stages of mitosis were acquired. A region was drawn around each cell to be measured, and the same size region was drawn in an area without fluorescent objects to be used for background subtraction. The net integrated intensity for each cell was measured at a single Z plane with highest integrated intensity values in the region of interest (this was usually the plane with the best focus). The weak signal from interphase cells was designated as 1, and the fluorescence intensity values at each mitotic stage were normalized and plotted relative to interphase. Each bar represents an average of 15–30 cells. The intensity of a signal from the control slide labeled with secondary antibodies alone was comparable to the intensity of the background in experimental samples.
