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Quiet periods were defined as periods in which the motion index changed by a rate of less than 0.025 arbitrary units per frame (with maximum rate of 1 given by the maximal pixel change) for at least 30 consecutive frames.
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Taking advantage of multiparameter and multispectral analysis provided by LSC iCys Research Imaging Cytometerr), which has three-color laser illumination and four channels of fluorescence measurement, the morphometric nuclear changes (maximal pixel and area) were measured in parallel with analysis of the induction of p21WAF1 and p27KIP1.
Thus, we studied the effect of exposure of A549 cells to one such inhibitor, TSA, by measuring the changes in intensity of maximal pixel DNA/DAPI fluorescence and nuclear area in the cells growing in its presence (Fig. 7).
The decrease in ratio of maximal pixel to nuclear area was even more sensitive senescence biomarker than the change in maximal pixel or nuclear area, each alone.
Thus, the biomarker based on analysis of nuclear area and intensity of maximal pixel of DNA-associated fluorescence is a sensitive reporter of the characteristic change in cell phenotype considered to be the hallmark of senescence.
The observed decrease in intensity of maximal pixel of DNA/DAPI fluorescence and the increase in nuclear area measured by LSC is a consequence of a change of cell geometry namely its extensive flattening of the senescent cell.
Since nuclear area in senescent cells is increased and DAPI maximal pixel decreased the ratio of maximal pixel to the area (Mp:area) provides an even more sensitive marker reporting this change in cell morphology than either of these measurements alone (Fig. 1B).
Background-corrected maximal pixel intensity (PImaxcorr) in the left ventricle was determined with videodensitometry.
The maximal pixel value in the raw projection data is five.
When the dilation was applied, the maximal pixel value overlapped by the structural element was found to replace the pixel value at the center of the structural element.
Maximal pixel amplitude within the tidal image was divided by 10, which results in 10 different amplitude categories according to which each pixel was then assigned.
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