High content image cytometry in the context of subnuclear organization

Vos, Winnok H. De; Neste, Leander Van; Dieriks, Birger; Joss, Greg; Oostveldt, Patric Van

doi:10.1002/cyto.a.20807

Cited by 65 publications

(49 citation statements)

References 65 publications

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“…For each sample, at least 12 fields were acquired on 5 z-stack focusses (1 µm). The γ-H2AX spot number and spot occupancy were analyzed with the INSCYDE plugin for ImageJ as previously described (21). Spot occupancy was defined for each nucleus as the sum of the spot areas divided by the nucleus area (spot_occupancy = sum (spot_area)/ nuclear_area).…”

Section: Dna Double-strand Break Detection (Detection Of γ-H2ax Foci)mentioning

confidence: 99%

Modulation of gene expression in endothelial cells in response to high LET nickel ion irradiation

Beck

Rombouts

Moreels

et al. 2014

International Journal of Molecular Medicine

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Abstract. Ionizing radiation can elicit harmful effects on the cardiovascular system at high doses. Endothelial cells are critical targets in radiation-induced cardiovascular damage. Astronauts performing a long-term deep space mission are exposed to consistently higher fluences of ionizing radiation that may accumulate to reach high effective doses. In addition, cosmic radiation contains high linear energy transfer (LET) radiation that is known to produce high values of relative biological effectiveness (RBE). The aim of this study was to broaden the understanding of the molecular response to high LET radiation by investigating the changes in gene expression in endothelial cells. For this purpose, a human endothelial cell line (EA.hy926) was irradiated with accelerated nickel ions (Ni) (LET, 183 keV/ µm) at doses of 0.5, 2 and 5 Gy. DNA damage was measured 2 and 24 h following irradiation by γ-H2AX foci detection by fluorescence microscopy and gene expression changes were measured by microarrays at 8 and 24 h following irradiation. We found that exposure to accelerated nickel particles induced a persistent DNA damage response up to 24 h after treatment. This was accompanied by a downregulation in the expression of a multitude of genes involved in the regulation of the cell cycle and an upregulation in the expression of genes involved in cell cycle checkpoints. In addition, genes involved in DNA damage response, oxidative stress, apoptosis and cell-cell signaling (cytokines) were found to be upregulated. An in silico analysis of the involved genes suggested that the transcription factors, E2F and nuclear factor (NF)-κB, may be involved in these cellular responses.

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Section: Dna Double-strand Break Detection (Detection Of γ-H2ax Foci)mentioning

confidence: 99%

Modulation of gene expression in endothelial cells in response to high LET nickel ion irradiation

Beck

Rombouts

Moreels

et al. 2014

International Journal of Molecular Medicine

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“…The kernel sizes for these operators are tunable parameters, which are automatically set to optimized values based on the image calibration (pixel size), retrieved from the metadata. In case of mitochondria, a normalized Laplace of Gaussian operator (LG) is applied, for which the optimal scale is automatically selected based on the most salient features in scale space (De Vos et al 2010). …”

Section: )mentioning

confidence: 99%

Integrated High-Content Quantification of Intracellular ROS Levels and Mitochondrial Morphofunction

Sieprath

Corne

Willems

et al. 2016

Advances in Anatomy, Embryology and Cell Biology

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Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-content microscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells. Principles of intracellular ROS generation and removalReactive oxygen species (ROS) are small, short-lived derivatives of molecular oxygen (O 2 ) of radical and non-radical nature (Halliwell and Gutteridge 2007 • ), nitrate radical (NO 3 • ) and many other nitrogen derivatives. ROS were originally described as molecular constituents of the defense system of phagocytic cells, but it has become clear that besides their damaging properties, they also function as signaling molecules and mediate a variety of other cellular responses including cell proliferation, differentiation, gene expression and migration (Lambeth 2004;Bartz and Piantadosi 2010). Intracellular ROS metabolismROS can be generated at various sites in the cell (Fig. 1A). This can be either deliberately, e.g. by NADPH oxidases (NOX), or as a byproduct, e.g. during normal cellular respiration in mitochondria (Babior 1999;Turrens 2003;Murphy 2009). The NOX family of NADPH oxidases (NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2) are proteins that transport electrons (e -) from NADPH across biological membranes (plasma or endomembranes) (Bedard and Krause 2007;Dupre-Crochet et al. 2013). The activation mechanisms and tissue distribution of the isoforms differ but they all use O 2 as e --acceptor, producing O 2•-. Through ROS generation, they play a role in many cellular processes including host defense, regulation of gene expression and cell differentiation (Bedard and Krause 2007). Despite their sometimes significant contribution to the global ROS pools, NOX are not the predominant source of intracellular ROS. Mitochondria are considered the major culprit, in particular under pathological conditions. Mitochondrial ROS are generated as a byproduct of the oxidative phosphorylation (OXPHOS, cf. below). Irrespective of its source, ROS production generally sta...

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“…As implemented in SynD 34 , particles with a unique local intensity maximum can be used to generate a datadriven single synapse kernel. Alternative solutions are multiscale spot segmentation approaches 72,73 or granulometric analysis to "sieve" image objects with structure elements based on their geometry and size 74 . In a final step, several criteria can be implemented for filtering false positive results.…”

Section: Counting Synaptic Punctamentioning

confidence: 99%

Image Informatics Strategies for Deciphering Neuronal Network Connectivity

Detrez

Verstraelen

Gebuis

et al. 2016

Focus on Bio-Image Informatics

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Brain function relies on an intricate network of highly dynamic neuronal connections that rewires dramatically under the impulse of various external cues and pathological conditions. Among the neuronal structures that show morphological plasticity are neurites, synapses, dendritic spines and even nuclei. This structural remodelling is directly connected with functional changes such as intercellular communication and the associated calcium-bursting behaviour. In vitro cultured neuronal networks are valuable models for studying these morpho-functional changes. Owing to the automation and standardisation of both image acquisition and image analysis, it has become possible to extract statistically relevant readout from such networks. Here, we focus on the current state-of-the-art in image informatics that enables quantitative microscopic interrogation of neuronal networks. We describe the major correlates of neuronal connectivity and present workflows for analysing them. Finally, we provide an outlook on the challenges that remain to be addressed, and discuss how imaging algorithms can be extended beyond in vitro imaging studies.2

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High content image cytometry in the context of subnuclear organization

Cited by 65 publications

References 65 publications

Modulation of gene expression in endothelial cells in response to high LET nickel ion irradiation

Modulation of gene expression in endothelial cells in response to high LET nickel ion irradiation

Integrated High-Content Quantification of Intracellular ROS Levels and Mitochondrial Morphofunction

Image Informatics Strategies for Deciphering Neuronal Network Connectivity

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