Relationship between chromatin compactness and dye uptake for in situ chromatin stained with DAPI

Mascetti, Giancarlo; Carrara, Sandro; Vergani, Laura

doi:10.1002/1097-0320(20010601)44:2<113::aid-cyto1089>3.0.co;2-a

Cited by 46 publications

(43 citation statements)

References 27 publications

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“…After permeabilization in 0.05% Triton X-100 solution (Sigma), ECs were stained with 25 µg ml − 1 DAPI and a stack of nuclear images at different focal positions along the z axis (0.15 µm intervals) were acquired in confocal scanning microscopy. To obtain quantitative information, shading correction and dark image substraction were applied in MATLAB to each image 38 and a constrained deconvolution in NIS Elements AR software was used to sharpen the image 56 . After normalizing for time exposure, the integrated fluorescence intensity was calculated as the sum of the intensity of each pixel.…”

Section: Methodsmentioning

confidence: 99%

“…After normalizing for time exposure, the integrated fluorescence intensity was calculated as the sum of the intensity of each pixel. The total fluorescence intensity was then divided by the nuclear volume to obtain the average spatial density, which correlates with the average chromatin packing ratio 56,57 , and as such is indicative of chromatin condensation. The proliferative activity in shape-engineered ECs has been investigated by detecting the incorporation of BrdU into cellular DNA.…”

Section: Methodsmentioning

confidence: 99%

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Spatial coordination between cell and nuclear shape within micropatterned endothelial cells

2012

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Growing evidence suggests that cytoplasmic actin filaments are essential factors in the modulation of nuclear shape and function. However, the mechanistic understanding of the internal orchestration between cell and nuclear shape is still lacking. Here we show that orientation and deformation of the nucleus are regulated by lateral compressive forces driven by tension in central actomyosin fibres. By using a combination of micro-manipulation tools, our study reveals that tension in central stress fibres is gradually generated by anisotropic force contraction dipoles, which expand as the cell elongates and spreads. our findings indicate that large-scale cell shape changes induce a drastic condensation of chromatin and dramatically affect cell proliferation. on the basis of these findings, we propose a simple mechanical model that quantitatively accounts for our experimental data and provides a conceptual framework for the mechanistic coordination between cell and nuclear shape.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Spatial coordination between cell and nuclear shape within micropatterned endothelial cells

2012

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“…Here we used the average 4′, 6-diamidino-2-phenylindole (DAPI) spatial density to assess global changes in chromatin condensation because the density of DAPI staining in nuclei increases with the chromatin packing ratio [36,37]. We found that S1 cells in 3D lrECM had higher DAPI density than cells cultured in 2D (Fig.…”

Section: Culturing Cells In 3d Lrecm Induces Alterations In Cellular mentioning

confidence: 99%

“…To assess the nuclear volume, a common intensity threshold was established to define the edge of the nuclei in each confocal section and calculate the enclosed area. The total fluorescence intensity was divided by the nuclear volume to obtain the average dye spatial density, which correlates with the average chromatin packing ratio (Mascetti et al 2001). All image processing was performed using Image J software.…”

Section: Immunofluorescence Analysismentioning

confidence: 99%

Cell shape regulates global histone acetylation in human mammary epithelial cells

Beyec

Lee

et al. 2007

Experimental Cell Research

150

120

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Extracellular matrix (ECM) regulates cell morphology and gene expression in vivo; these relationships are maintained in three-dimensional (3D) cultures of mammary epithelial cells. In the presence of laminin-rich ECM (lrECM), mammary epithelial cells round up and undergo global histone deacetylation, a process critical for their functional differentiation. However, it remains unclear whether lrECM-dependent cell rounding and global histone deacetylation are indeed part of a common physical-biochemical pathway. Using 3D cultures as well as nonadhesive and micropatterned substrata, here we showed that the cell 'rounding' caused by lrECM was sufficient to induce deacetylation of histones H3 and H4 in the absence of biochemical cues. Microarray and confocal analysis demonstrated that this deacetylation in 3D culture is associated with a global increase in chromatin condensation and a reduction in gene expression. Whereas cells cultured on plastic substrata formed prominent stress fibers, cells grown in 3D lrECM or on micropatterns lacked these structures. Disruption of the actin cytoskeleton with cytochalasin D phenocopied the lrECMinduced cell rounding and histone deacetylation. These results reveal a novel link between ECMcontrolled cell shape and chromatin structure, and suggest that this link is mediated by changes in the actin cytoskeleton.

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“…In the literature, there are many examples of use of CCD cameras for detecting fluorescent labelledDeoxyribonucleicAcid DNAsorsomeexpressions on the stained, fixed or live cells. Some examples to that is imaging of growing DNA chains [2], real-time detection of DNA hybridization to DNA microarrays [3], monitoring of anticancer effects of some specific agents [4], examining of cell polarity on stained, fixed and live cells [5] and obtaining quantitative information about the chromatin-DNA distribution inside the nucleus [6], [7], [8].…”

Section: Introductionmentioning

confidence: 99%

Empowering Low-Cost CMOS Cameras by Image Processing to Reach Comparable Results with Costly CCDs

et al. 2013

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Despite the hugeresearch effort to improve the performanceoftheComplementaryMetalOxideSemiconductor (CMOS) image sensors, Charge-Coupled Devices (CCDs) still dominate the cell biology related conventional fluorescence microscopic imaging market where low or ultra-low noise imaging is required. A detailed comparison of the sensor specifications and performance is usually not provided by the manufacturers which leads the end users not to go out of the habitude and choose a CCD camera instead of a CMOS one. However, depending on the application, CMOS cameras, when empowered by image processing algorithms can become cost-efficient solutions for conventional fluorescence microscopy. In this paper, we introduce an application-based comparative study between the default CCD camera of an inverted microscope (Nikon Ti-S Eclipse) and a custom-designed CMOS camera and apply efficient image processing algorithms to improve the performance of CMOS cameras. Quantum micro-bead samples that emit fluorescence light at different intensity levels, breast cancer diagnostic tissue cell and Caco-2 cell samples are imaged by both CMOS and CCD cameras and results are provided to show the reliability of CMOS camera processed images and finally to be of assistance when scientists select their cameras for desired applications.

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Relationship between chromatin compactness and dye uptake for in situ chromatin stained with DAPI

Cited by 46 publications

References 27 publications

Spatial coordination between cell and nuclear shape within micropatterned endothelial cells

Spatial coordination between cell and nuclear shape within micropatterned endothelial cells

Cell shape regulates global histone acetylation in human mammary epithelial cells

Empowering Low-Cost CMOS Cameras by Image Processing to Reach Comparable Results with Costly CCDs

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